Compounds for imaging Tau protein aggregates

ABSTRACT

The present invention relates to novel compounds of the formula (I) 
                         
that can be employed in the selective Tau detection of disorders and abnormalities associated with Tau aggregates such as Alzheimer&#39;s disease and other tauopathies using Positron Emission Tomography (PET) Imaging.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national stage of InternationalApplication No. PCT/EP2017/068500 filed on Jul. 21, 2017, and publishedin English under PCT Article 21(2), which claims the benefit of EuropeanPatent Application No. 16180905.8 filed Jul. 22, 2016. The entirecontents of the above-identified priority applications are hereby fullyincorporated herein by reference.

FIELD OF INVENTION

The present invention relates to novel compounds of the formula (I) thatcan be employed in the selective detection of disorders andabnormalities associated with Tau aggregates such as Alzheimer's disease(AD) and other tauopathies, for example, using Positron EmissionTomography (PET) imaging. The present invention also refers tointermediates which can be used in the production of such imagingcompounds. Diagnostic compositions as well as methods of imaging ordiagnosing using the above compounds and kits which are useful forpreparing a radiopharmaceutical preparation are also subject of thepresent invention.

BACKGROUND

Alzheimer's disease is a neurological disorder primarily thought to becaused by amyloid plaques, an extracellular accumulation of abnormaldeposit of amyloid-beta (Aβ) aggregates in the brain or in the eyes. Theother major neuropathological hallmarks in AD are the intracellularneurofibrillary tangles (NFT) that originate by the aggregation of thehyperphosphorylated Tau (Tubulin associated unit) protein,phosphorylated Tau or pathological Tau and its conformers. AD sharesthis pathology with many neurodegenerative tauopathies, in particularlywith specified types of frontotemporal dementia (FTD). In AD brain, Taupathology (tauopathy) develops later than amyloid pathology, but it isstill discussed controversially if Aβ protein is the causative agent inAD which constitutes the essence of the so-called amyloid cascadehypothesis (Hardy et al., Science 1992, 256, 184-185, and most recently,Musiek et al., Nature Neurosciences 2015, 18(6), 800-806, “Threedimensions of the amyloid hypothesis: time, space and ‘wingmen’”).

Presently, the only definite way to diagnose AD is to identify plaquesand tangles in brain tissue by histological analysis of biopsy orautopsy materials after the death of the individual. Beside AD, Tauplays an important role in other (non-AD) neurodegenerative diseases.Such non-AD tauopathies include, for example, supranuclear palsy (PSP),Pick's disease (PiD) and corticobasal degeneration (CBD).

Therefore, there is a great deal of interest in detection of Taupathology in vivo. Tau PET imaging promises novel insights intodeposition of Tau aggregates in the human brain and might allow tonon-invasively examine the degree of Tau pathology, quantify changes inTau deposition over time, assess its correlation with cognition andanalyze the efficacy of an anti-Tau therapy. For recent reviews see Shahet al., J Nucl Med. 2014, 55(6), 871-874: “Molecular Imaging Insightsinto Neurodegeneration: Focus on Tau PET Radiotracers”, Jovalekic etal., EJNMMI Radiopharmacy and Chemistry 2016, 1:11, “New proteindeposition tracers in the pipeline”, and Ariza et al., J Med Chem 2015,58(11), 4365-82: “Tau PET Imaging: Past, Present and Future”. Inaddition, several patent applications have recently been published, e.g:WO 2013/176698, WO 2009/102498, WO 2011/119565, U.S. Pat. No. 8,932,557B2 and U.S. Pat. No. 8,691,187, B2 (Siemens Medical Solutions, Lilly),WO 2012/067863 and WO 2012/068072 (both GE Healthcare) WO 2014/026881,WO 2014/177458, WO 2014/187762, WO 2015/044095, WO 2015/052105, WO2015/173225 (Hoffmann-La Roche AG), WO 2015/188368 (Merck Sharp & Dohme)and WO 2016/124508 (UCB Biopharma SPRL) which claim novel compounds forTau imaging.

In order to achieve high target selectivity, molecular probes have beenused which recognize and bind to the pathological target. Selectivityfor binding to pathological Tau protein over other protein depositionsin the brain is therefore a basic requirement of a Tau imaging probe. Inorder to reduce background signal interference resulting fromnon-specific off-target binding (e.g. binding to Aβ or monoamineoxidases), imaging compounds should bind with high affinity topathological Tau. Since amyloid or amyloid-like deposits formed fromproteins of diverse primary amino acid sequences share a common β-sheetquaternary conformation, molecular probes are required that candifferentiate such structures in order to avoid detection of otherpathologies (false-positives) and therefore misdiagnosis.

Off-target binding to monoamine oxidase A or B have been reported to bea significant limitation for Tau tracers, especially T-807 and THK-5351(Vermeiren, C, et al. Alzheimers & Dementia. 2015; 11 (7) Supplement p1-2: “T807, a reported selective tau tracer, binds with nanomolaraffinity to monoamine oxidase A”; Ng, K P, et al. Alzheimer's Researchand Therapy 2017, 9:25: “Monoamine oxidase B inhibitor, selegiline,reduces ¹⁸F-THK5351 uptake in the human brain”). Off-target binding tomonoamine oxidases A or B confound the interpretation of PET images withT807 and THK5351 with respect to tau. Presence of monoamine oxidaseswithin several brain regions limits the interpretation of PET imagingresults with these tracers.

Beside high selectivity, also binding to different Tau isoforms is animportant aspect for a tau tracer. Up till now, most tracers showbinding to tau in AD. However, tau in AD is a mixture of two isoforms,so called 3R-tau and 4R-tau. Other non-AD tauopathies are characterizedby the predominant presence of one of these isoforms. In Pick's disease(PiD), the 3R tau isoform is predominantly present whereas inprogressive supranuclear palsy (PSP) and in corticobasal degeneration(CBD), the 4R-tau isoform is the existing pathology.

In addition, molecular probes must also be designed such that uponadministration they can distribute within the body and reach theirtarget. For imaging of Tau aggregates associated with neurologicaldisorders such as e.g. Alzheimer's disease, imaging compounds arerequired that can penetrate the blood brain barrier and pass into therelevant regions of the brain. For targeting intracellular Tauaggregates, cell permeability is an additional requirement of imagingcompounds. A further prerequisite in order to get a sufficientsignal-to-noise ratio is a fast compound wash-out from non-targetregions in the brain (or other targeting organ). Also, compounds shouldshow no defluorination, as bone uptake in the skull (as result frompresence of free fluoride) will cause significant spill-over into thebrain which limits the usability (Chien D T, et al. J Alzheimers Dis.2014; 38:171-84).

The specifically disclosed and most advanced derivative of 2013/176698is 2,5-disubstituted pyridine compound ¹⁸F-1 (also see U.S. Pat. No.8,932,557 B2).

Compound ¹⁸F-1 was investigated in various clinical studies. Although¹⁸F-1 seems to be able to detect Tau in patients with AD or amyloid-betapositive mild cognitive impairment (MCI), various limitations have beenreported.

Vermeiren and coworkers found that compound ¹⁸F-1 bound to Monoamineoxidase A (MAO A) with a K_(D) of 1.5 nM. Their data unanimouslydemonstrate that compound ¹⁸F-1 binds to Tau aggregates and MAO-A withsimilar high affinity. The findings raise caution to the interpretationof compound ¹⁸F-1 clinical data, as MAO-A is widely expressed in mosthuman brain regions (Vermeiren et al., Alzheimers & Dementia. 2015; 11(7) Supplement p 1-2:T807-a reported selective Tau tracer, binds withnanomolar affinity to Monoamine oxidase A).

Compound ¹⁸F-1 was reported to have a fairly strong signal in parts ofthe brain's basal ganglia, e.g., the striatum and substantia nigra,regardless of the patient's diagnosis. The signal of ¹⁸F-1 in the cortexdid not reach a “steady state” (a window of time during which the ratioof binding in a target region to binding in the reference tissue (i.e.cerebellum) was stable). In addition, the kinetics of ¹⁸F-1 in variousbrain regions was different and never stabilized in a 150-minutescanning period (S. Baker, Human Amyloid Imaging Meeting, 2015).

Binding of compound ¹⁸F-1 to AD brain sections was demonstrated byautoradiography. However, compound ¹⁸F-1 showed limitations in bindingto brain sections with pathologies of non-AD tauopathies a) Lowe V J, etal. An autoradiographic evaluation of AV-1451 Tau PET in dementia. ActaNeuropathologica Communications. 2016; 4:58; b) Marquie M, et al.Validating novel Tau Positron Emission Tomography Tracer [F-18]-AV-1451(T807) on postmortem Brain Tissue. Annals of Neurology. 2015; 78:787; c)Gomez F, et al. Quantitative assessment of [¹⁸F]AV-1451 distribution inAD, PSP and PiD Post-Mortem Brain Tissue Sections relative to that ofthe anti-Tau antibody ATB. Journal of Nuclear Medicine. 2016; 57, S2:348, d) Sander K, et al. Characterization of tau positron emissiontomography tracer AV1451 binding to postmortem tissue in Alzheimer'sdisease, primary tauopathies, and other dementias. Alzheimers Dementia2016, 12(11): 116-1124 e) Smith R, et al. Increased basal gangliabinding of 18F-AV-1451 in patients with progressive supranuclear palsy.Movement disorders 2016.

Also clinically, ¹⁸F-1 seem to be of limited value for the detection oftau in PSP subjects a) Smith R et al., Tau neuropathology correlateswith FDG-PET, but nor with AV-1451-PET, in progressive supranuclearpalsy. Acta Neuropathologica 2017, 133:149-151; b) Smith R, et al.Increased basal ganglia binding of 18F-AV-1451 in patients withprogressive supranuclear palsy. Movement disorders 2017, 32(1), 108-114.

The final conclusions from these studies indicate that T807/AV1451 mightnot reliable to distinguish individual patients with PSP from controls.This is mainly attributed to an increased unspecific binding in midbrainstructures like basal ganglia. Uptake seen in cerebral cortex and whitematter did not reflected tau pathology in PSP.

Compound ¹⁸F-2 is disclosed in WO 2015/052105.

WO 2015/052105 only discloses one ¹⁸F-labeled compound and acorresponding compound which is tritium labeled. The compound comprisesa 2,5-disubstituted pyridine moiety (compound ¹⁸F-2). WO 2015/052105does not provide any data on binding to Tau-isoforms in non-ADtauopathies, binding to MAO A (or otherwise on selectivity to Tau),brain uptake, brain washout or retention in healthy brain, or any dataon in vivo de-fluorination.

¹⁸F-2 was found to not bind to brain tissue from patients with non-ADtauopathies such as Pick's disease (PiD) and progressive supranuclearpalsy (PSP) (Honer M et al., In vitro binding of ³H-RO6958948,³H-AV-1451, ³H-THK5351 and ³H-T808 to tau aggregates in non-ADtauopathies. Human Amyloid Imaging 2017, abstract 99).

In view of the above mentioned prior art, it was an object of thepresent invention to provide a compound which has a high affinity andselectivity for Tau and is thus suitable as a PET imaging agent.Preferably, the compounds of the present invention demonstrate highaffinity to Tau aggregates, high selectivity towards pathological Taucompared to other targets in the brain and favorable pharmacokineticproperties without defluorination.

SUMMARY OF THE INVENTION

Therefore, the present invention relates to the following items:

-   1. A compound of the formula (I)

-   -   as well as pharmaceutically acceptable salts, hydrates,        solvates, prodrugs and polymorphs thereof;    -   wherein    -   R¹ is selected from the group consisting of ¹⁸F, F and LG;    -   R² is H or PG;    -   PG is a protecting group;    -   LG is a leaving group.

-   2. The compound according to item 1, wherein R¹ is ¹⁸F and R² is H.

-   3. The compound according to item 1, wherein R¹ is F and R² is H.

-   4. The compound according to item 1, wherein R¹ is LG and R² is H or    PG.

-   5. The compound according to item 1 or 4, wherein R¹ is LG and R² is    H.

-   6. The compound according to item 1 or 4, wherein R¹ is LG and R² is    PG.

-   7. The compound according to item 1, 4, 5 or 6, wherein LG is nitro,    halogen or trimethyl ammonium.

-   8. The compound according to item 7, wherein LG is nitro or    trimethyl ammonium.

-   9. The compound according to item 1, 4, 6, 7, or 8, wherein PG is    tert-butyloxycarbonyl (BOC), triphenylmethyl (Trityl) or    dimethoxytrityl (DMT).

-   10. The compound according to item 9, wherein PG is    tert-butyloxycarbonyl (BOC).

-   11. The compound according to item 1, wherein the compound is    detectably labeled.

-   12. The compound according to item 11, wherein the detectable label    is selected from ²H, ³H and ¹⁸F.

-   13. The compound according to item 12, wherein the detectable label    is ¹⁸F.

-   14. A diagnostic composition comprising a compound as defined in any    of items 2, 11, 12 or 13 and optionally a pharmaceutically    acceptable carrier, diluent, adjuvant or excipient.

-   15. A compound as defined in item 2 or 13 for use in diagnostics.

-   16. A compound as defined in item 2 or 13 for use in the imaging of    Tau aggregates, particularly for use in positron emission tomography    imaging of Tau aggregates.

-   17. A compound as defined in item 2 or 13 for use in the diagnosis    of a disorder associated with Tau aggregates or for use in the    diagnosis of a tauopathy, particularly wherein the diagnosis is    conducted by positron emission tomography.

-   18. A compound for use according to item 17, wherein the tauopathy    is a 3R tauopathy.

-   19. A compound for use according to item 17, wherein the tauopathy    is a 4R tauopathy.

-   20. The compound for use according to item 17, wherein the disorder    is selected from Alzheimer's disease (AD), familial AD,    Creutzfeldt-Jacob disease, dementia pugilistica, Down's Syndrome,    Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis,    prion protein cerebral amyloid angiopathy, traumatic brain injury    (TBI), amyotrophic lateral sclerosis, Parkinsonism-dementia complex    of Guam, non-Guamanian motor neuron disease with neurofibrillary    tangles, argyrophilic grain disease, corticobasal degeneration    (CBD), diffuse neurofibrillary tangles with calcification,    frontotemporal dementia with Parkinsonism linked to chromosome 17,    Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick    disease type C, pallido-ponto-nigral degeneration, Pick's disease    (PiD), progressive subcortical gliosis, progressive supranuclear    palsy (PSP), subacute sclerosing panencephalitis, tangle only    dementia, postencephalitic Parkinsonism, myotonic dystrophy, Tau    panencephalopathy, AD-like with astrocytes, certain prion diseases    (GSS with Tau), mutations in LRRK2, chronic traumatic    encephalopathy, familial British dementia, familial Danish dementia,    frontotemporal lobar degeneration, Guadeloupean Parkinsonism,    neurodegeneration with brain iron accumulation, SLC9A6-related    mental retardation, white matter tauopathy with globular glial    inclusions, traumatic stress syndrome, epilepsy, Lewy body dementia    (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type),    mild cognitive impairment (MCI), multiple sclerosis, Parkinson's    disease, HIV-related dementia, adult onset diabetes, senile cardiac    amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary    retinal degeneration, macular degeneration (such as age-related    macular degeneration (AMD)), optic nerve drusen, optic neuropathy,    optic neuritis, and lattice dystrophy; preferably Alzheimer's    disease.

-   21. The compound for use according to item 20, wherein the disorder    is Alzheimer's disease (AD).

-   22. The compound for use according to item 20, wherein the disorder    is Parkinson's disease or atypical parkinsonism.

-   23. The compound for use according to item 20, wherein the disorder    is progressive supranuclear palsy (PSP).

-   24. The compound for use according to item 20, wherein the disorder    is Pick's disease (PiD).

-   25. The compound for use according to any one of items 16 to 24,    wherein the Tau aggregates are imaged in the brain or in the eye,    preferably wherein the detectable label is ¹⁸F and the imaging is    positron emission tomography.

-   26. A method of imaging of Tau aggregates, particularly a method of    positron emission tomography imaging of Tau aggregates, wherein an    effective amount of a compound as defined in item 2 or 13 is    administered to a patient.

-   27. A method of diagnosing a disorder associated with Tau aggregates    or a tauopathy, wherein an effective amount of a compound as defined    in item 2 or 13 is administered to a patient, particularly wherein    the diagnosis is conducted by positron emission tomography.

-   28. A method according to item 27, wherein the tauopathy is a 3R    tauopathy.

-   29. A method according to item 27, wherein the tauopathy is a 4R    tauopathy.

-   30. The method according to item 27, wherein the disorder is    selected from Alzheimer's disease (AD), familial AD,    Creutzfeldt-Jacob disease, dementia pugilistica, Down's Syndrome,    Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis,    prion protein cerebral amyloid angiopathy, traumatic brain injury,    amyotrophic lateral sclerosis, Parkinsonism-dementia complex of    Guam, non-Guamanian motor neuron disease with neurofibrillary    tangles, argyrophilic grain disease, corticobasal degeneration,    diffuse neurofibrillary tangles with calcification, frontotemporal    dementia with Parkinsonism linked to chromosome 17,    Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick    disease type C, pallido-ponto-nigral degeneration, Pick's disease,    progressive subcortical gliosis, progressive supranuclear palsy    (PSP), subacute sclerosing panencephalitis, tangle only dementia,    postencephalitic Parkinsonism, myotonic dystrophy, Tau    panencephalopathy, AD-like with astrocytes, certain prion diseases    (GSS with Tau), mutations in LRRK2, chronic traumatic    encephalopathy, familial British dementia, familial Danish dementia,    frontotemporal lobar degeneration, Guadeloupean Parkinsonism,    neurodegeneration with brain iron accumulation, SLC9A6-related    mental retardation, white matter tauopathy with globular glial    inclusions, traumatic stress syndrome, epilepsy, Lewy body dementia    (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type),    mild cognitive impairment (MCI), multiple sclerosis, Parkinson's    disease, HIV-related dementia, adult onset diabetes, senile cardiac    amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary    retinal degeneration, macular degeneration (such as age-related    macular degeneration (AMD)), optic nerve drusen, optic neuropathy,    optic neuritis, and lattice dystrophy; preferably Alzheimer's    disease.

-   31. The method according to item 30, wherein the disorder is    Alzheimer's disease (AD).

-   32. The method according to item 30, wherein the disorder is    Parkinson's disease or atypical parkinsonism.

-   33. The method according to item 30, wherein the disorder is    progressive supranuclear palsy (PSP).

-   34. The method according to item 30, wherein the disorder is Pick's    disease (PiD).

-   35. The method according to any one of items 26 to 34, wherein the    Tau aggregates are imaged in the brain or in the eye, preferably    wherein the detectable label is ¹⁸F and the imaging is positron    emission tomography.

-   36. Use of a compound as defined in item 2 or 13 for the manufacture    of a diagnostic agent for imaging of Tau aggregates, particularly    for positron emission tomography imaging of Tau aggregates.

-   37. Use of a compound as defined in item 2 or 13 for the manufacture    of a diagnostic agent for diagnosing a disorder associated with Tau    aggregates or for diagnosing a tauopathy, particularly wherein the    diagnosis is conducted by positron emission tomography.

-   38. The use according to item 37, wherein the tauopathy is a 3R    tauopathy.

-   39. The use according to item 37, wherein the tauopathy is a 4R    tauopathy.

-   40. The use according to item 37, wherein the disorder is selected    from Alzheimer's disease (AD), familial AD, Creutzfeldt-Jacob    disease, dementia pugilistica, Down's Syndrome,    Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis,    prion protein cerebral amyloid angiopathy, traumatic brain injury,    amyotrophic lateral sclerosis, Parkinsonism-dementia complex of    Guam, non-Guamanian motor neuron disease with neurofibrillary    tangles, argyrophilic grain disease, corticobasal degeneration,    diffuse neurofibrillary tangles with calcification, frontotemporal    dementia with Parkinsonism linked to chromosome 17,    Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick    disease type C, pallido-ponto-nigral degeneration, Pick's disease,    progressive subcortical gliosis, progressive supranuclear palsy    (PSP), subacute sclerosing panencephalitis, tangle only dementia,    postencephalitic Parkinsonism, myotonic dystrophy, Tau    panencephalopathy, AD-like with astrocytes, certain prion diseases    (GSS with Tau), mutations in LRRK2, chronic traumatic    encephalopathy, familial British dementia, familial Danish dementia,    frontotemporal lobar degeneration, Guadeloupean Parkinsonism,    neurodegeneration with brain iron accumulation, SLC9A6-related    mental retardation, white matter tauopathy with globular glial    inclusions, traumatic stress syndrome, epilepsy, Lewy body dementia    (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutch type),    mild cognitive impairment (MCI), multiple sclerosis, Parkinson's    disease, HIV-related dementia, adult onset diabetes, senile cardiac    amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary    retinal degeneration, macular degeneration (such as age-related    macular degeneration (AMD)), optic nerve drusen, optic neuropathy,    optic neuritis, and lattice dystrophy; preferably Alzheimer's    disease.

-   41. The use according to item 40, wherein the disorder is    Alzheimer's disease (AD).

-   42. The use according to item 40, wherein the disorder is    Parkinson's disease or atypical parkinsonism.

-   43. The use according to item 40, wherein the disorder is    progressive supranuclear palsy (PSP).

-   44. The use according to item 40, wherein the disorder is Pick's    disease (PiD).

-   45. The use according to any one of items 36 to 44, wherein the Tau    aggregates are imaged in the brain or in the eye, preferably wherein    the detectable label is ¹⁸F and the imaging is positron emission    tomography.

-   46. Use of the compound according to item 3 as an analytical    reference.

-   47. Use of the compound according to item 3 as an in vitro screening    tool.

-   48. A method of preparing a compound as defined in item 2 comprising    reacting a compound as defined in item 4 with a [¹⁸F]fluorinating    agent, wherein the method further comprises cleaving of the    protecting group PG, if present.

-   49. The method according to item 48, wherein the [¹⁸F]fluorinating    agent is selected from K¹⁸F, H¹⁸F, Cs¹⁸F, Na¹⁸F and a tetra(C₁₋₆    alkyl) ammonium salt of ¹⁸F.

-   50. A method of preparing a diagnostic composition as defined in    item 14 comprising reacting a compound as defined in item 4 with a    [¹⁸F]fluorinating agent, wherein the method further comprises    cleaving of the protecting group PG, if present, and subsequently    optionally admixing a pharmaceutically acceptable carrier, diluent,    adjuvant or excipient.

-   51. A kit for preparing a radiopharmaceutical preparation, said kit    comprising a sealed vial containing a predetermined quantity of a    compound as defined in item 4.

-   52. The kit according to item 51, which further comprises at least    one component selected from a reaction solvent, a solid-phase    extraction cartridge, a reagent for cleaving the protecting group, a    solvent for purification, a solvent for formulation and a    pharmaceutically acceptable carrier, diluent, adjuvant or excipient    for formulation.

-   53. A method of collecting data for the diagnosis of a disorder    associated with tau aggregates in a sample or a patient comprising:    -   (a) bringing a sample or a specific body part or body area        suspected to contain a tau aggregate into contact with a        compound as defined in items 11 to 13;    -   (b) allowing the compound to bind to the tau aggregate;    -   (c) detecting the compound bound to the tau aggregate; and    -   (d) optionally correlating the presence or absence of compound        binding with the tau aggregate with the presence or absence of        tau aggregate in the sample or specific body part or body area.

-   54. A method of determining the amount of tau aggregate in a tissue    and/or a body fluid comprising:    -   (a) providing a sample representative of the tissue and/or body        fluid under investigation;    -   (b) testing the sample for the presence of tau aggregate with a        compound as defined in items 11 to 13;    -   (c) determining the amount of compound bound to the tau        aggregate; and    -   (d) calculating the amount of tau aggregate in the tissue and/or        body fluid.

-   55. A method of collecting data for determining a predisposition to    a disorder associated with tau aggregates in a patient comprising    detecting the specific binding of a compound as defined in items 11    to 13 to a tau aggregate in a sample or in situ which comprises the    steps of:    -   (a) bringing the sample or a specific body part or body area        suspected to contain the tau aggregate into contact with the        compound as defined in items 11 to 13, which compound        specifically binds to the tau aggregate;    -   (b) allowing the compound to bind to the tau aggregate to form a        compound/tau aggregate complex;    -   (c) detecting the formation of the compound/tau aggregate        complex;    -   (d) optionally correlating the presence or absence of the        compound/tau aggregate complex with the presence or absence of        tau aggregate in the sample or specific body part or body area;        and    -   (e) optionally comparing the amount of the compound/tau        aggregate to a normal control value.

-   56. A method of collecting data for monitoring residual disorder in    a patient suffering from a disorder associated with tau aggregates    who has been treated with a medicament, wherein the method    comprises:    -   (a) bringing a sample or a specific body part or body area        suspected to contain a tau aggregate into contact with a        compound as defined in items 11 to 13, which compound        specifically binds to the tau aggregate;    -   (b) allowing the compound to bind to the tau aggregate to form a        compound/tau aggregate complex;    -   (c) detecting the formation of the compound/tau aggregate        complex;    -   (d) optionally correlating the presence or absence of the        compound/tau aggregate complex with the presence or absence of        tau aggregate in the sample or specific body part or body area;        and    -   (e) optionally comparing the amount of the compound/tau        aggregate to a normal control value.

-   57. A method of collecting data for predicting responsiveness of a    patient suffering from a disorder associated with tau aggregates and    being treated with a medicament comprising:    -   (a) bringing a sample or a specific body part or body area        suspected to contain an tau aggregate into contact with a        compound as defined in items 11 to 13, which compound        specifically binds to the tau aggregate;    -   (b) allowing the compound to bind to the tau aggregate to form a        compound/tau aggregate complex;    -   (c) detecting the formation of the compound/tau aggregate        complex;    -   (d) optionally correlating the presence or absence of the        compound/tau aggregate complex with the presence or absence of        tau aggregate in the sample or specific body part or body area;        and    -   (e) optionally comparing the amount of the compound/tau        aggregate to a normal control value.

It is understood that the present invention covers compounds of theformula (I) in which one or more of the respective atoms is replaced bya different isotope. For instance, the compounds of the formula (I)include compounds in which one or more of the hydrogen atoms is replacedby tritium and/or one or more of the hydrogen atoms is replaced bydeuterium.

The present inventors have surprisingly found that the compounds of theformula (I) in which R¹ is ¹⁸F or F and R² is H (compounds F-4 and¹⁸F-4, respectively) have significantly improved properties compared tothe prior art compounds ¹⁸F-1 or ¹⁸F-2.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Autoradiography of AD and HC brain slices with compound ¹⁸F-4.In the AD brain sections, a strong punctated staining was detectablethat could be blocked with the addition of excess corresponding coldcompound. In the healthy control (HC) sections, no specific signal wasvisible.

FIG. 2: Washout curves depicting the clearance of the activity from thenormal brain for compounds ¹⁸F-1, ¹⁸F-2 and ¹⁸F-4.

FIG. 3: Brain up-take and wash-out of ¹⁸F-4 in a non-demented humancontrol subject.

FIG. 4: a)¹⁸F-4 PET image of a non-demented human control subject withaxial, sagittal and coronal projection, b)¹⁸F-4 PET image of an ADsubject with axial, sagittal and coronal projection.

DETAILED DESCRIPTION

The present invention relates to detectably labeled compounds of theformula (I)

that can be employed in the selective detection of disorders andabnormalities associated with Tau aggregates such as Alzheimer's diseaseand other tauopathies, for example, by using Positron EmissionTomography (PET) imaging. The present invention also refers tointermediates which can be used in the production of such imagingcompounds. The present compounds have a high affinity for Tau and bindto Tau-isoforms present in both, Alzheimer's disease (AD), as well as innon-AD tauopathies, such as for example progressive supranuclear palsy(PSP), and Pick's disease (PiD). Since they have a low affinity foramyloid-beta and MAO A they can be used as highly selective molecularprobes for binding pathological Tau and thus avoid detection of otherpathologies and misdiagnosis.

The instant ¹⁸F-labeled compounds also lead to a low signal in healthybrain, so that they can reduce background signal interference and thusprovide a low detection limit.

Due to their good brain uptake, fast washout from healthy brain, lowlong-term retention in healthy brain as well as the lack of in vivode-fluorination the instant compounds provide a good signal-to-noiseratio.

Furthermore, the instant compounds can be easily detectably labeled,e.g., with ¹⁸F, in high yields.

Definitions

The term “protecting group” (PG) as employed herein is any protectinggroup which is suitable for protecting an amine group during anenvisaged chemical reaction. Examples of suitable protecting groups arewell-known to a person skilled in the art. Suitable protecting groupsare discussed, e.g., in the textbook Greene and Wuts, Protecting groupsin Organic Synthesis, third edition, page 494-653, which is includedherein by reference. Protecting groups can be chosen from carbamates,amides, imides, N-alkyl amines, N-aryl amines, imines, enamines,boranes, N—P protecting groups, N-sulfenyl, N-sulfonyl and N-silyl.Specific preferred examples of protecting groups (PG) are carbobenzyloxy(Cbz), p-methoxybenzyl carbonyl (Moz or MeOZ), tert-butyloxycarbonyl(BOC), 9-fluorenylmethyloxycarbonyl (FMOC), benzyl (Bn), p-methoxybenzyl(PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP),triphenylmethyl (Trityl), methoxyphenyl diphenylmethyl (MMT), ordimethoxytrityl (DMT). More preferred examples of the protecting groupPG include tert-butyloxycarbonyl (BOC), dimethoxytrityl (DMT) andtriphenylmethyl (Trityl). One more preferred example of the protectinggroup PG is tert-butyloxycarbonyl (BOC).

The term “leaving group” (LG) as employed herein is any leaving groupand means an atom or group of atoms can be replaced by another atom orgroup of atoms. Examples are given e.g. in Synthesis (1982), p. 85-125,table 2, Carey and Sundberg, Organische Synthese, (1995), page 279-281,table 5.8; or Netscher, Recent Res. Dev. Org. Chem., 2003, 7, 71-83,scheme 1, 2, 10 and 15 and others). (Coenen, Fluorine-18 LabelingMethods: Features and Possibilities of Basic Reactions, (2006), in:Schubiger P. A., Friebe M., Lehmann L., (eds), PET-Chemistry—The DrivingForce in Molecular Imaging. Springer, Berlin Heidelberg, pp. 15-50,explicitly: scheme 4 pp. 25, scheme 5 pp 28, table 4 pp 30, FIG. 7 pp33). Preferably, the “leaving group” (LG) is nitro, halogen or trimethylammonium. More preferably, “leaving group” (LG) is nitro or trimethylammonium. In one preferred embodiment, “leaving group” (LG) is nitro. Inanother preferred embodiment, “leaving group” (LG) is trimethylammonium.

Tau as used herein refers to a highly soluble microtubule bindingprotein mostly found in neurons and includes the major 6 isoforms,cleaved or truncated forms, and other modified forms such as arisingfrom phosphorylation, glycosylation, glycation, prolyl isomerization,nitration, acetylation, polyamination, ubiquitination, sumoylation andoxidation. Pathologic Tau or Tau aggregates (Neurofibrillary Tangles,NFTs) as used herein refer to insoluble aggregates of thehyperphosphorylated Tau protein containing paired helical filaments andstraight filaments. Their presence is a hallmark of AD and otherdiseases known as tauopathies.

The term “polymorphs” refers to the various crystalline structures ofthe compounds of the present invention. This may include, but is notlimited to, crystal morphologies (and amorphous materials) and allcrystal lattice forms. Salts of the present invention can be crystallineand may exist as more than one polymorph.

Solvates, hydrates as well as anhydrous forms of the present compoundsare also encompassed by the invention. The solvent included in thesolvates is not particularly limited and can be any pharmaceuticallyacceptable solvent. Examples include water and C₁₋₄ alcohols (such asmethanol or ethanol).

As used hereinafter in the description of the invention and in theclaims, the term “prodrug” means any covalently bonded compound whichreleases the active parent pharmaceutical due to in vivobiotransformation. The reference by Goodman and Gilman (ThePharmacological Basis of Therapeutics, 8 ed, McGraw-Hill, Int. Ed. 1992,“Biotransformation of Drugs”, p 13-15) describing prodrugs generally ishereby incorporated herein by reference.

As used hereinafter in the description of the invention and in theclaims, the term “pharmaceutically acceptable salt” relates to non-toxicderivatives of the disclosed compounds wherein the parent compound ismodified by making salts of inorganic and organic acids thereof.Inorganic acids include, but are not limited to, acids such ascarboxylic, hydrochloric, nitric or sulfuric acid. Organic acidsinclude, but are not limited to, acids such as aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic andsulphonic acids. The pharmaceutically acceptable salts of the presentinvention can be synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two. Lists ofsuitable salts can be found in Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Company, Easton, Pa., 1990, p. 1445, the disclosureof which is hereby incorporated by reference.

“Pharmaceutically acceptable” is defined as those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication commensurate with areasonable benefit/risk ratio.

The patients or subjects in the present invention are typically animals,particularly mammals, more particularly humans.

The tau gene contains 16 exons with the major tau protein isoforms beingencoded by 11 of them The alternative splicing of exon 10 generates tauisoforms with either three (exon 10 missing) or four (exon 10 present)repeat domains, known as 3R and 4R tau, respectively (A. Andreadis etal., Biochemistry 31, (1992) 10626-10633; M. Tolnay et al., IUBMB Life,55(6): 299-305, 2003). In Alzheimer's disease, the ratio of 3R and 4Risoforms is similar. In contrast thereto, in some tauopathies one of thetwo isoforms is predominantly present. Herein, the term “3R tauopathy”refers to tauopathies (such as Pick's disease (PiD)) in which the 3Risoform is predominantly present. Herein, the term “4R tauopathy” refersto tauopathies (such as progressive supranuclear palsy (PSP) andcorticobasal degeneration (CBD)) in which the 4R isoform ispredominantly present.

The preferred definitions given in the “Definition”-section apply to allof the embodiments described herein unless stated otherwise.

Diagnostic Procedures

The detectably labeled compounds of the present invention (in particular¹⁸F-4) are particularly suitable for imaging of Tau protein aggregates.With respect to Tau protein, the detectably labeled compounds of thepresent invention (in particular ¹⁸F-4) are able to bind to varioustypes of Tau aggregates such as pathologically aggregated Tau,hyperphosphorylated Tau, neurofibrillary tangles, paired helicalfilaments, straight filaments, neurotoxic soluble oligomers, polymersand fibrils.

Due to the above binding characteristics, the detectably labeledcompounds of the present invention (in particular ¹⁸F-4) are suitablefor use in the diagnosis of disorders associated with Tau aggregates.The detectably labeled compounds of the present invention (in particular¹⁸F-4) are particularly suitable for positron emission tomography (PET)imaging of Tau deposits. Typically ¹⁸F labeled compounds of the formula(I) are employed as detectably labeled compounds if the compounds are tobe administered to a patient.

In the imaging of Tau aggregates a detectably labeled compound of theformula (I) (preferably ¹⁸F-4) is administered and the signal stemmingfrom the compound that is specifically bound to the Tau aggregates isdetected. The specific binding is a result of the high binding affinityof the compounds of the formula (I) to the Tau aggregates.

In a preferred embodiment, a detectably labeled compound of the formula(I) (preferably ¹⁸F-4) is employed for diagnosing whether a tauopathy(preferably Alzheimer's disease) is present. In this method a detectablylabeled compound of the formula (I) (preferably ¹⁸F-4) is administeredto a patient who is suspected to suffer from a tauopathy (preferablyAlzheimer's disease) or a sample obtained from such a patient and thesignal stemming from the detectable label is detected, preferably bypositron emission tomography (PET).

If no signal stemming from the detectable label is detected then theinstant method can be used to exclude a tauopathy, which indicates thata neurological disorder other than a tauopathy is present.

In the methods of diagnosing a disorder associated with Tau proteinaggregates such as Alzheimer's disease, or a predisposition therefor ina subject, the method comprising:

-   a) administering to the mammal a diagnostically effective amount of    a detectably labeled compound of the present invention (in    particular ¹⁸F-4);-   b) allowing the detectably labeled compound of the present invention    (in particular ¹⁸F-4) to distribute into the tissue of interest    (such as brain tissue, the eye or body fluids such as cerebrospinal    fluid (CSF)); and-   c) imaging the tissue of interest, wherein an increase in binding of    the detectably labeled compound of the present invention (in    particular ¹⁸F-4) to the tissue of interest compared to a normal    control level of binding indicates that the subject is suffering    from or is at risk of developing a disorder associated with Tau    protein aggregates.

The detectably labeled compounds of the present invention (in particular¹⁸F-4) can be used for imaging of Tau protein aggregates in any sampleor a specific body part or body area of a patient which suspected tocontain a Tau protein aggregate. The detectably labeled compounds of thepresent invention (in particular ¹⁸F-4) are able to pass the blood-brainbarrier and to pass into the eye. Consequently, they are particularlysuitable for imaging of Tau protein aggregates in the brain, in the eye(ophthalmic and/or retinal imaging) as well as in body fluids such ascerebrospinal fluid (CSF).

In diagnostic applications, the detectably labeled compounds of thepresent invention (in particular ¹⁸F-4) are preferably administered in adiagnostic composition.

Diagnosis of a Tau disorder or of a predisposition to a Tau-associateddisorder in a patient may be achieved by detecting the specific bindingof a detectably labeled compound of the present invention (in particular¹⁸F-4) to the Tau protein aggregates in a sample or in situ, whichincludes:

-   (a) bringing the sample or a specific body part or body area    suspected to contain the Tau protein aggregate into contact with a    detectably labeled compound of the present invention (in particular    ¹⁸F-4) which binds the Tau protein aggregate;-   (b) allowing the detectably labeled compound of the present    invention (in particular ¹⁸F-4) to bind to the Tau protein aggregate    to form a compound/Tau protein aggregate complex (hereinafter    “compound/Tau protein aggregate complex” will be abbreviated as    “compound/protein aggregate complex”);-   (c) detecting the formation of the compound/protein complex,-   (d) optionally correlating the presence or absence of the    compound/protein complex with the presence or absence of Tau protein    aggregates in the sample or specific body part or area; and-   (e) optionally comparing the amount of the compound/protein to a    normal control value, wherein an increase in the amount of the    compound/protein compared to a normal control value may indicate    that the patient is suffering from or is at risk of developing a    Tau-associated disorder.

After the sample or a specific body part or body area has been broughtinto contact with the detectably labeled compound of the presentinvention (in particular ¹⁸F-4), the compound is allowed to bind to theTau protein aggregate. The amount of time required for binding willdepend on the type of test (e.g., in vitro or in vivo) and can bedetermined by a person skilled in the field by routine experiments.

The compound which has bound to the Tau protein aggregate can besubsequently detected by any appropriate method. A preferred method ispositron emission tomography (PET).

The presence or absence of the compound/protein is then optionallycorrelated with the presence or absence of Tau protein aggregates in thesample or specific body part or area. Finally, the amount of thecompound/protein can be compared to a normal control value which hasbeen determined in a sample or a specific body part or body area of ahealthy subject, wherein an increase in the amount of thecompound/protein compared to a normal control value may indicate thatthe patient is suffering from or is at risk of developing aTau-associated disorder.

The present invention also relates to a method of determining the amountof Tau protein aggregate in a tissue and/or a body fluid. This methodcomprises the steps of:

-   (a) providing a sample representative of the tissue and/or body    fluid under investigation;-   (b) testing the sample for the presence of Tau protein aggregate    with a detectably labeled compound of the present invention (in    particular ¹⁸F-4);-   (c) determining the amount of the detectably labeled compound of the    present invention (in particular ¹⁸F-4) bound to the Tau protein    aggregate; and-   (d) calculating the amount of Tau protein aggregate in the tissue    and/or body fluid.

The sample can be tested for the presence of Tau protein aggregate witha detectably labeled compound of the present invention (in particular¹⁸F-4) by bringing the sample into contact with a detectably labeledcompound of the present invention (in particular ¹⁸F-4), allowing thedetectably labeled compound of the present invention (in particular¹⁸F-4) to bind to the Tau protein aggregate to form a compound/proteinaggregate complex and detecting the formation of the compound/proteincomplex as explained above.

Monitoring minimal residual disorder in a patient suffering from adisorder associated with Tau protein aggregates who has been treatedwith a medicament with a detectably labeled compound of the presentinvention (in particular ¹⁸F-4) may be achieved by:

-   (a) bringing a sample or a specific body part or body area suspected    to contain a Tau protein aggregate into contact with a detectably    labeled compound of the present invention (in particular ¹⁸F-4);-   (b) allowing the detectably labeled compound of the present    invention (in particular ¹⁸F-4) to bind to the Tau protein aggregate    to form a compound/protein aggregate complex;-   (c) detecting the formation of the compound/protein aggregate    complex;-   (d) optionally correlating the presence or absence of the    compound/protein aggregate complex with the presence or absence of    Tau protein aggregate in the sample or specific body part or body    area; and-   (e) optionally comparing the amount of the compound/protein    aggregate to a normal control value, wherein an increase in the    amount of the aggregate compared to a normal control value may    indicate that the patient may still suffer from a minimal residual    disease.

How steps (a) to (e) can be conducted has already been explained above.

Predicting responsiveness of a patient suffering from a disorderassociated with Tau protein aggregates and being treated with amedicament can be achieved by

-   (a) bringing a sample or a specific body part or body area suspected    to contain a Tau protein aggregate into contact with a detectably    labeled compound of the present invention (in particular ¹⁸F-4);-   (b) allowing the detectably labeled compound of the present    invention (in particular ¹⁸F-4) to bind to the Tau protein aggregate    to form a compound/protein aggregate complex;-   (c) detecting the formation of the compound/protein aggregate    complex;-   (d) optionally correlating the presence or absence of the    compound/protein aggregate complex with the presence or absence of    Tau protein aggregate in the sample or specific body part or body    area; and-   (e) optionally comparing the amount of the compound/protein    aggregate to a normal control value.

How steps (a) to (e) can be conducted has already been explained above.

In the method for predicting responsiveness the amount of thecompound/protein complex can be optionally compared at various points oftime during the treatment, for instance, before and after onset of thetreatment or at various points of time after the onset of the treatment.A change, especially a decrease, in the amount of the compound/proteincomplex may indicate that the patient has a high potential of beingresponsive to the respective treatment.

A compound according to the present invention can also be incorporatedinto a test kit for detecting a Tau protein aggregate. The test kittypically comprises a container holding one or more compounds accordingto the present invention and instructions for using the compound for thepurpose of binding to a Tau protein aggregate to form a compound/proteincomplex and detecting the formation of the compound/protein complex suchthat presence or absence of the compound/protein complex correlates withthe presence or absence of the Tau protein aggregates.

The term “test kit” refers in general to any diagnostic kit known in theart. More specifically, the latter term refers to a diagnostic kit asdescribed in Zrein et al., Clin. Diagn. Lab. Immunol., 1998, 5, 45-49.

Diagnostic Compositions

A “diagnostic composition” is defined in the present invention as acomposition comprising a detectably labeled compound of the presentinvention (preferably ¹⁸F labeled; in particular ¹⁸F-4). For in vivoapplications the diagnostic composition should be in a form suitable foradministration to mammals such as humans. Preferably a diagnosticcomposition further comprises a physiologically acceptable carrier,diluent, adjuvant or excipient. Administration to a patient ispreferably carried out by injection of the composition as an aqueoussolution. Such a composition may optionally contain further ingredientssuch as solvents, buffers; pharmaceutically acceptable solubilizers; andpharmaceutically acceptable stabilizers or antioxidants.

Pharmaceutically acceptable excipients are well known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, 15^(th) Ed., Mack Publishing Co., New Jersey(1975). The pharmaceutical excipient can be selected with regard to theintended route of administration and standard pharmaceutical practice.The excipient must be acceptable in the sense of being not deleteriousto the recipient thereof.

Pharmaceutically useful excipients that may be used in the formulationof the diagnostic composition of the present invention may comprise, forexample, carriers, vehicles, diluents, solvents and edible oils, oilyesters, binders, adjuvants, solubilizers, thickening agents,stabilizers, disintegrants, glidants, lubricating agents, bufferingagents, emulsifiers, wetting agents, suspending agents, sweeteningagents, colorants, flavors, coating agents, preservatives, antioxidants,processing agents, drug delivery modifiers and enhancers.

If the detectably labeled compounds of the present invention (preferably¹⁸F labeled, in particular ¹⁸F-4) are administered parenterally, thenexamples of such administration include one or more of: intravenously,intraarterially, intraperitoneally, intrathecally, intraventricularly,intraurethrally, intrasternally, intracranially, intramuscularly orsubcutaneously administering the compounds; and/or by using infusiontechniques. For parenteral administration, the compounds are best usedin the form of a sterile aqueous solution which may contain otherexcipients. The aqueous solutions should be suitably buffered(preferably to a pH of from 3 to 9), if necessary. The preparation ofsuitable parenteral formulations under sterile conditions is readilyaccomplished by standard pharmaceutical techniques well known to thoseskilled in the art.

The dose of the detectably labeled compounds of the present invention(preferably ¹⁸F labeled, in particular ¹⁸F-4) will vary depending on theexact compound to be administered, the weight of the patient, size andtype of the sample, and other variables as would be apparent to aphysician skilled in the art. Generally, the dose could preferably liein the range 0.001 μg/kg to 10 μg/kg, preferably 0.01 μg/kg to 1.0μg/kg. The radioactive dose can be, e.g., 100 to 600 MBq, morepreferably 150 to 450 MBq.

The diagnostic compositions of the invention can be produced in a mannerknown per se to the skilled person as described, for example, inRemington's Pharmaceutical Sciences, 15^(th) Ed., Mack Publishing Co.,New Jersey (1975).

For instance, the compounds of the present invention can be employed ina liposomal composition as described in WO2016057812A1 which comprises acompound of formula (II) as a ligand for use in the selective detectionof disorders and abnormalities associated with Tau aggregates bynonradioactive magnetic resonance imaging (MRI).

In particular, in one embodiment diseases or disorders that can bedetected and monitored with the detectably labeled compounds of thepresent invention (in particular ¹⁸F-4) are diseases or conditionsassociated Tau proteins aggregates.

The diseases or conditions that can be detected and monitored with thedetectably labeled compounds of the present invention (in particular¹⁸F-4) include neurodegenerative disorders such as tauopathies. Examplesof diseases and conditions which can be detected and monitored arecaused by or associated with the formation of neurofibrillary lesions.This is the predominant brain pathology in tauopathy. The diseases andconditions comprise a heterogeneous group of neurodegenerative diseasesor conditions including diseases or conditions which show co-existenceof Tau and amyloid pathologies. Examples of diseases involving Tauaggregates are generally listed as tauopathies and these include, butare not limited to, Alzheimer's disease (AD), Creutzfeldt-Jacob disease,dementia pugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinkerdisease, inclusion-body myositis, prion protein cerebral amyloidangiopathy, traumatic brain injury, amyotrophic lateral sclerosis,Parkinsonism-dementia complex of Guam, non-Guamanian motor neurondisease with neurofibrillary tangles, argyrophilic grain disease,corticobasal degeneration, diffuse neurofibrillary tangles withcalcification, frontotemporal dementia with Parkinsonism linked tochromosome 17, Hallervorden-Spatz disease, multiple system atrophy,Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Pick'sdisease, progressive subcortical gliosis, progressive supranuclear palsy(PSP), subacute sclerosing panencephalitis, tangle only dementia,postencephalitic Parkinsonism, myotonic dystrophy, Taupanencephalopathy, AD-like with astrocytes, certain prion diseases (GSSwith Tau), mutations in LRRK2, chronic traumatic encephalopathy,familial British dementia, familial Danish dementia, frontotemporallobar degeneration, Guadeloupean Parkinsonism, neurodegeneration withbrain iron accumulation, SLC9A6-related mental retardation, white mattertauopathy with globular glial inclusions, traumatic stress syndrome,epilepsy, Lewy body dementia (LBD), hereditary cerebral hemorrhage withamyloidosis (Dutch type), mild cognitive impairment (MCI), multiplesclerosis, Parkinson's disease, HIV-related dementia, adult onsetdiabetes, senile cardiac amyloidosis, endocrine tumors, glaucoma, ocularamyloidosis, primary retinal degeneration, macular degeneration (such asage-related macular degeneration (AMD)), optic nerve drusen, opticneuropathy, optic neuritis, and lattice dystrophy. Preferably thediseases and conditions which can be detected and monitored includeAlzheimer's disease (AD), familial AD, Creutzfeldt-Jacob disease,dementia pugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinkerdisease, inclusion-body myositis, prion protein cerebral amyloidangiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis,Parkinsonism-dementia complex of Guam, non-Guamanian motor neurondisease with neurofibrillary tangles, argyrophilic grain disease,corticobasal degeneration (CBD), diffuse neurofibrillary tangles withcalcification, frontotemporal dementia with Parkinsonism linked tochromosome 17, Hallervorden-Spatz disease, multiple system atrophy,Niemann-Pick disease type C, pallido-ponto-nigral degeneration, Pick'sdisease (PiD), progressive subcortical gliosis, progressive supranuclearpalsy (PSP), subacute sclerosing panencephalitis, tangle only dementia,postencephalitic Parkinsonism, myotonic dystrophy, Taupanencephalopathy, AD-like with astrocytes, certain prion diseases (GSSwith Tau), mutations in LRRK2, chronic traumatic encephalopathy,familial British dementia, familial Danish dementia, frontotemporallobar degeneration, Guadeloupean Parkinsonism, neurodegeneration withbrain iron accumulation, SLC9A6-related mental retardation, and whitematter tauopathy with globular glial inclusions, more preferablyAlzheimer's disease (AD), Creutzfeldt-Jacob disease, dementiapugilistica, amyotrophic lateral sclerosis, argyrophilic grain disease,corticobasal degeneration, frontotemporal dementia with Parkinsonismlinked to chromosome 17, Pick's disease, progressive supranuclear palsy(PSP), tangle only dementia, Parkinson dementia complex of Guam,Hallervorden-Spatz disease and fronto-temporal lobar degeneration.Preferably the disease or condition is Alzheimer's disease.

General Synthesis of ¹⁸F-Labeled Compounds of the Present Invention

Compounds having the formula (I) which are labeled by ¹⁸F can beprepared by reacting a compound of formula (I), in which R¹ is LG and R²is H or PG, with an ¹⁸F-fluorinating agent, so that the leaving group LGis replaced by ¹⁸F. The preparation includes the cleavage of theprotecting group PG, if present.

Any suitable ¹⁸F-fluorinating agent can be employed. Typical examplesinclude H¹⁸F, alkali or alkaline earth ¹⁸F-fluorides (e.g., K¹⁸F, Rb¹⁸F,Cs¹⁸F, and Na¹⁸F). Optionally, the ¹⁸F-fluorination agent can be used incombination with a chelating agent such as a cryptand (e.g.:4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane—Kryptofix®)or a crown ether (e.g.: 18-crown-6). Alternatively, the ¹⁸F-fluorinatingagent can be a tetraalkyl ammonium salt of ¹⁸F or a tetraalkylphosphonium salt of ¹⁸F; e.g., tetra(C₁₋₆ alkyl)ammonium salt of ¹⁸F ora tetra(C₁₋₆ alkyl)phosphonium salt of ¹⁸F. Examples thereof includetetrabutyl ammonium [¹⁸F]fluoride and tetrabutyl phosphonium[¹⁸F]fluoride. Preferably, the ¹⁸F-fluorination agent is K¹⁸F, H¹⁸F,Cs¹⁸F, Na¹⁸F or tetrabutyl ammonium [¹⁸F]fluoride.

The reagents, solvents and conditions which can be used for the¹⁸F-fluorination are well-known to a person skilled in the field (L.Cai, S. Lu, V. Pike, Eur. J. Org. Chem 2008, 2853-2873; J. FluorineChem., 27 (1985): 177-191; Coenen, Fluorine-18 Labeling Methods:Features and Possibilities of Basic Reactions, (2006), in: Schubiger P.A., Friebe M., Lehmann L., (eds), PET-Chemistry—The Driving Force inMolecular Imaging. Springer, Berlin Heidelberg, pp. 15-50). Preferably,the solvents used in the ¹⁸F-fluorination are DMF, DMSO, acetonitrile,DMA, or mixtures thereof, preferably the solvent is acetonitrile orDMSO.

If desired, the compound having the formula (I) can have R¹ is LG and R²is PG, wherein the protecting group PG protects the amine during the¹⁸F-fluorination reaction. This amine protecting group can besubsequently removed. Methods for removing the amine protecting groupare known in the art and include, but are not limited to, acidiccleavage.

If desired, the compound of formula (I) can be isolated and/or purifiedfurther before use. Corresponding procedures are well-known in the art.

The precursor compounds having the formula (I) in which R¹ is LG and R²is H or PG can be provided in a kit which is suitable for producing thecompounds of the formula (I) by reaction with a ¹⁸F-fluorinating agent.In one embodiment the kit comprises a sealed vial containing apredetermined quantity of the precursor compound of the presentinvention. For instance, the kit can contain 1.5 to 75 μmol, preferably7.5 to 50 μmol, more preferably 10 to 30 μmol of a precursor compound(I) of the present invention. Optionally, the kit can contain furthercomponents, such as a reaction solvent, a solid-phase extractioncartridge, a reagent to obtain the ¹⁸F-fluorinating agent, a reagent forcleaving the protecting group, a solvent for purification, a solvent forformulation and a pharmaceutically acceptable carrier, diluent, adjuvantor excipient for formulation.

The compounds of the present invention in which R¹ is F and R² is H canbe used as an analytical reference or an in vitro screening tool.

The compounds of the present invention in which R¹ is F and R² is H canbe used as an analytical reference for the quality control and releaseof a compound of the present invention in which R¹ is ¹⁸F and R² is H.

The compounds of the present invention in which R¹ is F and R² is H canbe used as an in vitro screening tool for characterization of tissuewith Tau pathology and for testing of compounds targeting Tau pathologyon such tissue.

The present invention illustrated by the following examples which shouldnot be construed as limiting.

EXAMPLES

All reagents and solvents were obtained from commercial sources and usedwithout further purification. Proton (¹H) spectra were recorded on aBruker DRX-400 MHz NMR spectrometer or on a Bruker AV-400 MHz NMRspectrometer in deuterated solvents. Mass spectra (MS) were recorded onan Advion CMS mass spectrometer. Chromatography was performed usingsilica gel (Fluka: Silica gel 60, 0.063-0.2 mm) and suitable solvents asindicated in the specific examples. Flash purification was conductedwith a Biotage Isolera One flash purification system using HP-Sil(Biotage) or puriFlash-columns (Interchim) and the solvent gradientindicated in the specific examples. Thin layer chromatography (TLC) wascarried out on silica gel plates with UV detection.

Although some of the present examples do not indicate that therespective compounds were detectably labeled, it is understood thatcorresponding detectably labeled compounds can be easily prepared, e.g.,by using detectably labeled starting materials, such as startingmaterials containing ³H atoms.

ABBREVIATIONS

AD Alzheimer's disease Boc, BOC tert-butyloxycarbonyl CBD corticobasaldegeneration d.c. corrected for decay d doublet dd doublet of doubletddd doublet of doublet of doublet dt doublet of triplet DMF N,N-dimethylformamide DMSO dimethylsulfoxide EI electron ionisation ELSD evaporativelight scattering detector ESI electrospray ionisation FTD Frontotemporaldementia HPLC high performance liquid chromatography HC Healthy controlGBq Gigabequerel K₂₂₂ 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane (Kryptofix 222) MBq Megabequerel MS mass spectrometry MeCNacetonitrile m multiplet mc centered multiplet n.c.a. non-carrier-addedn.d.c. not decay corrected NMR nuclear magnetic resonance spectroscopy:chemical shifts (δ) are given in ppm. PET Positron-Emission-TomographyPiD Pick's disease PSP progressive supranuclear palsy q quadruplet(quartet) RT room temperature s singulet t triplet Tau Tau protein, Taudeposits, Tau aggregates TBI Traumatic brain injury Trt trityl(triphenylmethyl) TLC thin layer chromatography

Preparative Example A

Step A

Commercially available 2,6-dibromopyridine (4.12 g, 16.6 mmol) wassuspended in ethanol (40 mL) and hydrazine hydrate (10 mL, 97.6 mmol) inwater (˜50-60%) was added. The mixture was heated in a sand-bath at˜115° C. for 18 hours. The solvent was removed and the residue waspurified by chromatography on silica using ethyl acetate/n-heptane(60/40) to afford the title compound as an off-white solid (3.05 g,93%).

¹H-NMR (400 MHz, CDCl₃): δ=7.33 (t, 1H), 6.83 (d, 1H), 6.67 (d, 1H),6.00 (br-s, 1H), 3.33-3.00 (br-s, 2H)

Step B

The title compound from Step A above (10 g, 53.2 mmol) and commerciallyavailable 1-Boc-4-piperidone (10.6 g, 53.2 mmol) were added to a 500 mLflask and mixed to become a homogenous blend. Then polyphosphoric acid(80 g, 115% H₃PO₄ basis) was added and the mixture was heated at ˜160°C. in a sand-bath. At ˜120° C. the Boc-protecting group was cleavedresulting in foaming of the reaction mixture. After completeBoc-cleavage the foam collapsed and the dark reaction mixture wasstirred at ˜160° C. for 20 hours. The reaction was allowed to cool toroom temperature and water (400 mL) was added. The reaction mixture wasstirred/sonicated until the gummy material was dissolved. The reactionmixture was then placed in an ice-bath and the pH of the solution wasadjusted to pH ˜12 by adding solid sodium hydroxide pellets(exothermic). The precipitate was collected by filtration and washedwith water (400 mL) to remove salts. The precipitate was dissolved indichloromethane/methanol (9/1; 1500 mL) by sonication and washed withwater (2×400 mL) to remove remaining salts and insoluble material. Theorganic phase was dried over Na₂SO₄, filtered and the solvents wereremoved under reduced pressure. The dark residue was treated withdichloromethane (100 mL), sonicated for 5 minutes and the precipitatewas collected by filtration. The precipitate was washed withdichloromethane (40 mL) and air-dried to afford the title compound abeige solid (3.5 g, 26%).

¹H-NMR (400 MHz, DMSO-d₆): δ=11.5 (br-s, 1H), 7.72 (d, 1H), 7.15 (d,1H), 3.86-3.82 (m, 2H), 3.06-3.00 (m, 2H), 2.71-2.65 (m, 2H)

Step C

The title compound from Step B above (1.75 g, 6.94 mmol) was suspendedin xylene (380 mL) and manganese (IV) oxide (6.62 g, 76.9 mmol) wasadded. The reaction mixture was then heated at ˜160° C. in a sand-bathfor 36 hours. The cooled reaction mixture was evaporated under reducedpressure, the residue suspended in dichloromethane/methanol (1/1; 400mL) and stirred at room temperature for 30 minutes. The reaction mixturewas then filtered through paper filters to remove the manganese (IV)oxide and the filter washed with methanol (50 mL). The combinedfiltrates were evaporated under reduced pressure and the dark residuepurified by chromatography on silica (50 g HP-SIL-cartridge) using aBiotage Isolera system employing an ethyl acetate/heptane gradient(5/95-100/0) to remove unpolar impurities followed bydichloromethane/methanol (9/1→4/1) to afford the title compound as darkyellow solid. The total yield from 2 runs was 1.77 g (51%).

¹H-NMR (400 MHz, DMSO-d₆): δ=12.52 (br-s, 1H), 9.42 (s, 1H), 8.61 (d,1H), 8.53 (d, 1H), 7.56-7.52 (m, 2H)

Preparative Example B

Step A

To a suspension of the title compound from Preparative Example A (0.776g, 3.13 mmol) in dichloromethane (65 mL) was added triethylamine (1.86mL, 13 mmol) and trityl-chloride (2.63 g, 9.39 mmol). After the additionof 4-(dimethylamino)-pyridine (0.074 g, 0.608 mmol), the reactionmixture was stirred at room temperature for 16 hours. The reactionmixture was diluted with dichloromethane (150 mL) and water (50 mL). Theorganic phase was separated, dried over Na₂SO₄, filtered and thesolvents removed in vacuo. The residue was purified on HP-Sil SNAPcartridges (50 g) using a Biotage Isolera One purification systememploying an ethyl acetate/n-heptane gradient (5/95→100/0→100/0) toafford the title compound B as pale yellow solid (0.831 g, 54%).Unreacted starting material was recovered by flushing the cartridge withethyl acetate/methanol (90/10) to afford the starting material asoff-white solid (0.195 g, 25%).

¹H-NMR (400 MHz, CDCl₃) δ=9.22 (s, 1H), 8.23 (d, 1H), 8.13 (d, 1H),7.48-7.42 (m, 7H), 7.33-7.22 (m, 12H), 6.41 (d, 1H)

MS (ESI); m/z=490.03/491.96 [M+H]⁺

Example 1

Step A

To a mixture of degassed 1,4-dioxane (4.3 mL) and water (1 mL) in amicrowave vial was added[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (0.0084 g, 0.01 mmol), followed by the titlecompound from Preparative Example A (0.05 g, 0.2 mmol),(2-fluoropyridin-3-yl)boronic acid (0.035 g, 0.245 mmol) and cesiumcarbonate (0.133 g, 0.41 mmol). The reaction mixture was then heated at˜110° C. in a sand-bath for 16 hours. The reaction mixture was dilutedwith ethyl acetate (60 mL) and water (20 mL), the organic phase wasseparated, dried over Na₂SO₄, filtered and the solvents were evaporatedin vacuo. The dark residue was purified by chromatography on silica (10g HP-SIL) using a Biotage Isolera system employing adichloromethane/methanol gradient (100/0→95/5→90/10) to afford a mixtureof the title compound and unreacted starting material. The mixture wasfurther purified by two times preparative TLC (500 μM Analtech Uniplate(20×20 cm)) using dichloromethane/methanol (9/1) as mobile phase toafford the title compound F-4 as a white solid (0.0039 g, 7.3%).

¹H NMR (400 MHz, DMSO-d₆) δ=12.45 (br-s, 1H), 8.78 (d, 1H), 8.62-8.52(m, 2H), 8.37-8.35 (m, 1H), 7.84 (dd, 1H), 7.61-7.57 (m, 1H), 7.57 (d,1H)

MS (ESI): m/z=265.01 [M+H]⁺

Example 2

Step A

In a 5 ml microwave tube was dissolved the title compound fromPreparative Example A (0.05 g, 0.202 mmol) and(2-fluoropyridin-3-yl)boronic acid (0.0568 g, 0.403 mmol) inN,N′-dimethylacetamide (Volume: 2.015 mL). Sodium carbonate 2 M (0.403mL, 0.806 mmol) was added and the resulting stirring solution wasdegassed for 5 minutes.[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex withdichloromethane was added (0.017 g, 0.02 mmol) and the reaction mixturewas heated to 110° C. for 3 hours. TLC monitoring showed completion ofthe reaction. The reaction mixture was diluted with ethyl acetate, afterinsolubles were filtered out, the filtrate was washed with water andbrine. The organic layer was dried with MgSO₄, filtered andconcentrated. The residue was purified by chromatography on silica (10 gHP-SIL) using a Biotage Isolera system employing adichloromethane/methanol gradient (100/0→90/10) to afford the titlecompound F-4 as a beige solid (0.028 g, 54%).

¹H-NMR (400 MHz, DMSO-d₆) δ=12.45 (s, 1H), 9.43 (s, 1H), 8.77 (d, 1H),8.66-8.45 (m, 2H), 8.35 (d, 1H), 7.83 (dd, 1H), 7.64-7.39 (m, 2H)

MS (ESI); m/z=265.19 [M+H]⁺

Example 3

Step A

To a mixture of degassed 1,4-dioxane (8 mL) in a microwave vial wasadded [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II),complex with dichloromethane (0.034 g, 0.04 mmol), followed by the titlecompound from Preparative Example B (0.2 g, 0.4 mmol),bis(pinacolato)diborane (0.112 g, 0.44 mmol) and potassium acetate(0.118 g, 1.2 mmol). The reaction mixture was then heated at ˜95° C. ina sand-bath for 18 hours. The reaction mixture was diluted with ethylacetate (100 mL) and water (30 mL), the organic phase separated, driedover Na₂SO₄, filtered and the solvents evaporated in vacuo to afford thecrude title compound which was directly used in the next step.

Step B

The crude title compound from Step A above was dissolved in a mixture ofdegassed 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave vial. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II), complexwith dichloromethane (0.034 g, 0.04 mmol), 3-bromo-2-nitropyridine (0.1g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol) were added andthe reaction mixture was heated at ˜115° C. in a sand-bath for 6 hours.

The reaction mixtures of 4 runs as described in Steps A and B werecombined and diluted with ethyl acetate (600 mL) and water (200 mL), theorganic phase separated, dried over Na₂SO₄, filtered and the solventsevaporated in vacuo. The dark residue was split in two portions and eachportion was purified by chromatography on silica (40 g BGB-column,Interchim) using a Biotage Isolera system employing an ethylacetate/n-heptane gradient (5/95→100/0→100/0) to afford a mixture of themore polar title compound and a less polar byproduct. The mixture (0.260g) was further purified by preparative TLC with a loading of ˜0.03 g ofmixture per 1000 μM Analtech Uniplate (20×20 cm) using ethylacetate/n-heptane (70/30) as mobile phase to afford the more polar titlecompound 14 as pale yellow solid (0.1 g, 11.8%).

More polar title compound 14:

¹H NMR (400 MHz, CDCl₃) δ=9.28 (s, 1H), 8.48-8.46 (m, 1H); 8.38 (d, 1H),8.26 (d, 1H), 7.55-7.53 (m, 1H). 7.52-7.46 (m, 6H), 7.32-7.20 (m, 11H),6.46 (d, 1H)

MS (ESI): m/z=534.17 [M+H]⁺.

Example 4

Step A

The title compound from Example 3 (compound 14; 0.0434 g, 0.082 mmol)was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1.2mL) was added. The reaction mixture was stirred at room temperature for6 hours. The reaction mixture was diluted with diluted withdichloromethane (50 mL) and water (20 mL). The pH of the aqueous phasewas adjusted to pH-12 by the addition of a 1 M aqueous sodium hydroxidesolution. The aqueous layer was separated, extracted withdichloromethane (25 mL), the combined organic layer dried over Na₂SO₄,filtered and the solvent removed under reduced pressure. The residue waspurified by chromatography on silica (10 g HP-SIL-column) using aBiotage Isolera system employing a dichloromethane/methanol gradient(100/0→95/5→90/10→80/20) to afford the title compound as an off-whitesolid (0.0199 g, 83%)

¹H NMR (400 MHz, DMSO-d₆) δ=12.45 (br-s, 1H), 9.45 (s, 1H), 8.85 (s,1H), 8.67 (dd, 1H), 8.58 (dd, 1H), 8.54 (d, 1H), 7.97 (dd, 1H); 7.82 (d,1H), 7.50 (d, 1H)

MS (ESI): m/z=291.81 [M+H]⁺.

Example 5

Step A

To a solution of the title compound from Example 3 (compound 14; 0.038g, 0.071 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid(1.2 mL). The reaction mixture was stirred at room temperature for 6hours and methanol (2 mL) was added. The solvents were evaporated invacuo and the residue dissolved/suspended in methanol (5 mL). Thesolvents were evaporated in vacuo and the residue dissolve/suspended inmethanol (5 mL). The solvents were evaporated in vacuo and the residuesuspended in dichloromethane (2 mL). After the addition of triethylamine(1 mL, 7.2 mmol), di-tert-butyl dicarbonate (0.098 g, 0.43 mmol), and4-(dimethylamino)-pyridine (0.0018 g, 0.014 mmol), the reaction mixturewas stirred at room temperature for 18 hours. The reaction mixture wasdiluted with ethyl acetate (50 mL) and water (20 mL). The organic phasewas separated, dried aver Na₂SO₄, filtered and the solvents removed invacuo. The residue was purified on silica (25 g puriFlash, Interchim)using a Biotage Isolera One purification system employing an ethylacetate/n-heptane gradient (5/95→100/0→100/0) to afford the titlecompound as pale yellow solid (0.0184 g, 66%).

¹H-NMR (400 MHz, CDCl₃) δ=9.34 (s, 1H), 8.75 (d, 1H), 8.65-8.62 (m, 1H),8.50 (d, 1H), 8.33 (d, 1H); 8.26 (d, 1H), 7.76-7.72 (m, 1H), 7.62 (d,1H), 1.77 (s, 9H) MS (ESI); m/z=392.33 [M+H]⁺

Comparative Example 2 (F-2)

Step A

To a suspension of the title compound from Preparative Example A (0.430g, 1.73 mmol) in dichloromethane (25 mL) was added triethylamine (1.93mL, 13.89 mmol) and di-tert-butyl dicarbonate (2.27 g, 10.02 mmol).After the addition of 4-(dimethylamino)-pyridine (0.042 g, 0.34 mmol),the reaction mixture was stirred at room temperature for 3 days. Thesolvents were removed under reduced pressure and the residue waspurified on HP-Sil SNAP cartridges (25 g) using a Biotage Isolera Onepurification system employing an ethyl acetate/n-heptane gradient(5/95→100/0→100/0) to afford the title compound as off-white solid(0.558 g, 92%).

¹H-NMR (400 MHz, CDCl₃) δ=9.28 (s, 1H), 8.73 (d, 1H), 8.22 (d, 2H), 7.598d, 1H), 1.80 (s, 9H)

Step B

To a mixture of degassed 1,4-dioxane (3 mL) and water (0.7 mL) in amicrowave vial was added[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (0.0058 g, 0.007 mmol), followed by the titlecompound from Step A above (0.05 g, 0.143 mmol),(6-fluoropyridin-3-yl)boronic acid (0.024 g, 0.17 mmol) and cesiumcarbonate (0.092 g, 0.286 mmol). The reaction mixture was then heated at˜100° C. in a sand-bath for 4 hours. The reaction mixture was dilutedwith ethyl acetate (80 mL) and water (35 mL), the organic phaseseparated, dried over Na₂SO₄, filtered and the solvents evaporated invacuo. The dark residue was purified by chromatography on silica (12 g,puriFlash, Interchim) using a Biotage Isolera system employing adichloromethane/methanol gradient (100/0→98/2→95/5→90/10→80/20) toafford the less polar Boc-protected compound (0.0255 g, 49%) and themore polar Comparative Example C2 (F-2) as off-white solid (0.0116 g,31%).

More Polar Comparative Example C2 (F-2):

¹H NMR (400 MHz, DMSO-d₆) δ=12.40 (br-s, 1H), 9.40 (s, 1H), 9.05 (s,1H), 8.78-8.70 (m, 2H), 8.51 (d, 1H), 8.02 (d, 1H), 7.50 (d, 1H), 7.36(dd, 1H)

MS (ESI): m/z=265.09 [M+H]⁺

Less Polar Boc-Protected Compound:

¹H NMR (400 MHz, DMSO-d₆) δ=9.48 (s, 1H), 9.13 (d, 1H), 8.84-8.78 (m,2H), 8.68 (d, 1H), 8.23 (d, 1H), 8.19 (d, 1H), 7.40 (dd, 1H), 1.75 8s,9H)

The synthesis of Comparative Example C2 (F-2) was first described inWO2015/052105 (Example 1) by a different synthesis.

Comparative Example 2 (F-2) Precursor

Step A

To a mixture of degassed 1,4-dioxane (3 mL) and water (0.7 mL) in amicrowave vial was added[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (0.0058 g, 0.007 mmol), followed by the titlecompound from Comparative Example 2 Step A (0.05 g, 0.143 mmol),2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (0.0428g, 0.17 mmol) and cesium carbonate (0.092 g, 0.286 mmol). The reactionmixture was then heated at ˜100° C. in a sand-bath for 4 hours. Thereaction mixture was diluted with ethyl acetate (80 mL) and water (35mL), the organic phase separated, dried over Na₂SO₄, filtered and thesolvents evaporated in vacuo. The dark residue was purified bychromatography on silica (12 g, puriFlash, Interchim) using a BiotageIsolera system employing a dichloromethane/methanol gradient(100/0→98/2→95/5→90/10→80/20) to afford the Comparative Example C2 (F-2)Precursor as a pale yellow solid (0.0173 g, 31%).

¹H NMR (400 MHz, CDCl₃/CD₃OD) δ=9.45 (d, 1H), 9.32 (s, 1H), 8.93 (dd,1H), 8.68-8.64 (m, 2H), 8.46 (d, 1H), 8.35 (d, 1H), 8.14 (d, 1H), 1.82(s, 9H)

MS (ESI): m/z=392.13 [M+H]⁺

The synthesis of Comparative Example C2 (F-2) precursor was firstdescribed in WO2015/052105 (Example 3a) by a different synthesis.

Comparative Example 5 (F-5)

Step A

To a mixture of degassed 1,4-dioxane (4.3 mL) and water (1 mL) in amicrowave vial was added[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (0.0084 g, 0.01 mmol), followed by the titlecompound from Preparative Example A (0.05 g, 0.2 mmol),3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (0.055g, 0.246 mmol) and cesium carbonate (0.133 g, 0.41 mmol). The reactionmixture was then heated at ˜115° C. in a sand-bath for 6 hours. Thereaction mixture was diluted with ethyl acetate (60 mL) and water (20mL), the organic phase separated, dried over Na₂SO₄, filtered and thesolvents evaporated in vacuo. The dark residue was purified bychromatography on silica (25 g HP-SIL) using a Biotage Isolera systememploying a dichloromethane/methanol gradient (100/0→95/5→90/10→80/20)to afford the Comparative Example C5 (F-5) as off-white solid (0.022 g,43%).

¹H NMR (400 MHz, DMSO-d₆) δ=12.45 (br-s, 1H), 9.45 (s, 1H), 9.31 (s,1H), 8.80 (d, 1H), 8.67 (d, 1H). 8.53 (d, 1H), 8.46-8.40 (m, 1H), 8.11(d, 1H), 7.52 (d, 1H) MS (ESI): m/z=265.06 [M+H]⁺

Comparative Example 5 (F-5) Precursor

Step A

To a mixture of degassed 1,4-dioxane (4 mL) in a microwave vial wasadded [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II),complex with dichloromethane (0.017 g, 0.02 mmol), followed by the titlecompound from Preparative Example B (0.1 g, 0.2 mmol),bis(pinacolato)diborane (0.056 g, 0.22 mmol) and potassium acetate(0.059 g, 0.6 mmol). The reaction mixture was then heated at ˜95° C. ina sand-bath for 18 hours. The reaction mixture was diluted with ethylacetate (100 mL) and water (30 mL), the organic phase separated, driedover Na₂SO₄, filtered and the solvents evaporated in vacuo to afford thecrude title compound which was directly used in the next step.

Step B

The crude title compound from Step A above was dissolved in a mixture ofdegassed 1,4-dioxane (4.3 mL) and water (1 mL) in a microwave vial. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II), complexwith dichloromethane (0.017 g, 0.02 mmol), 3-bromo-5-nitropyridine (0.05g, 0.245 mmol) and cesium carbonate (0.133 g, 0.41 mmol) were added andthe reaction mixture was heated at ˜115° C. in a sand-bath for 6 hours.

The reaction mixture was diluted with ethyl acetate (80 mL) and water(30 mL), the organic phase separated, dried over Na₂SO₄, filtered andthe solvents evaporated in vacuo. The dark residue was purified bychromatography on silica (25 g puriFlash, Interchim) using a BiotageIsolera system employing an ethyl acetate/n-heptane gradient(5/95→100/0→100/0) to afford the Comparative Example C5 (F-5) Precursoras pale yellow solid (0.0144 g, 13%).

¹H NMR (400 MHz, CDCl₃) δ=9.36 (d, 1H), 9.30 (s, 1H), 9.02 (d, 1H);8.52-8.48 (m, 2H), 8.29 (d, 1H), 7.80 (d, 1H), 7.60-7.55 (m, 5H),7.33-7.25 (m, 10H), 6.46 (d, 1H) MS (ESI): m/z=533.67 [M+H]⁺.

Comparative Example 6 (F-6)

Step A

In a 20 ml microwave tube was dissolved the title compound fromPreparative Example B (0.2 g, 0.408 mmol) and4-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (0.182g, 0.816 mmol) in N,N′-dimethylacetamide (5.10 mL). Sodium carbonate(0.816 ml, 1.631 mmol) was added and the resulting stirring solution wasdegassed for 5 minutes. Then[1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex withdichloromethane was added and the reaction mixture was heated to 110° C.for 22 hours. TLC monitoring showed completion of the reaction. Thereaction mixture was diluted with dichloromethane, insolubles werefiltered out through Celite, and the filtrate was washed with waterthree times to remove residual amounts of N,N′-dimethylacetamide. Theorganic layer was dried MgSO₄, filtered and concentrated. The residuewas purified via Biotage Isolera One (100:0 to 90:10dichloromethane/methanol; 25 g HP-Sil column) to give afford the titlecompound (0.1036 g; 50%).

¹H-NMR (400 MHz, DMSO-d₆) δ=9.43 (s, 1H), 8.75 (d, 1H), 8.54 (dd, 1H),8.26 (d, 1H), 8.17 (d, 1H), 7.83 (dd, 1H), 7.61-7.52 (m, 6H), 7.41 (dd,1H), 7.35-7.20 (m, 9H), 6.46 (d, 1H).

MS [M+H]⁺=507.43, 243.29

Step B

In a 25 ml round bottom flask, was dissolved the title compound fromStep A above (0.1 g, 0.199 mmol) in dichloromethane (1 mL).Trifluoroacetic acid (1 mL) was carefully added and the reaction mixturewas stirred for 18 hours at room temperature. After cooling at 0° C.,the reaction mixture was quenched to pH=10 with 2 M sodium hydroxidesolution. The resulting suspension was filtered. The reaction mixturewas washed with water and brine. The organic was dried over MgSO₄,filtered and concentrated. The residue was purified via Biotage IsoleraOne (100:0 to 90:10 dichloromethane/methanol; 10 g HP-Sil column) togive afford the Comparative Example C6 (F-6) (0.026 g; 47%).

¹H-NMR (400 MHz, DMSO-d₆) δ 12.57 (s, 1H), 9.46 (s, 1H), 9.19 (d, 1H),8.80 (d, 1H), 8.70 (s, 1H), 8.59-8.52 (m, 1H), 7.81 (d, 1H), 7.55 (d,2H)

MS [M+H]⁺=265.29

Comparative Example 6 (F-6) Precursor

In a 20 ml microwave tube was dissolved the title compound fromPreparative Example B (0.2 g, 0.408 mmol) and4-nitro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (0.204g, 0.816 mmol) in N,N′-dimethylacetamide (5.10 mL). Sodium carbonate(0.816 ml, 1.631 mmol) was added and the resulting stirring solution wasdegassed for 5 minutes. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex withdichloromethane was added (0.017 g, 0.02 mmol) and the reaction mixturewas heated to 110° C. for 22 hours. TLC monitoring showed completion ofthe reaction. The reaction mixture was diluted with dichloromethane,insolubles were filtered out through Celite, and the filtrate was washedwith water three times to remove residual amounts ofN,N′-dimethylacetamide. The organic layer was dried MgSO₄, filtered andconcentrated. The residue was purified via Biotage Isolera One employingan ethyl acetate/n-heptane gradient (5/95→100/0→100/0) to afford theComparative Example C6 (F-6) Precursor as pale yellow solid (0.056 g,28%).

¹H-NMR (400 MHz, DMSO-d₆) δ=9.45 (s, 1H), 8.81 (d, 1H), 8.69 (d, 1H),8.32-8.23 (m, 3H), 8.20 (d, 1H), 7.60 (dd, 6H), 7.36-7.22 (m, 9H), 6.52(d, 1H), 5.76 (s, 1H).

MS (ESI): m/z=533.87 [M+H]⁺.

Comparative Example 7 (F-7)

Step A

To a mixture of degassed 1,4-dioxane (4.3 mL) and water (1 mL) in amicrowave vial was added[1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex withdichloromethane (0.0084 g, 0.01 mmol), followed by the title compoundfrom Preparative Example A (0.05 g, 0.2 mmol),2-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (0.055g, 0.246 mmol) and cesium carbonate (0.133 g, 0.41 mmol). The reactionmixture was then heated at ˜115° C. in a sand-bath for 6 hours. Thereaction mixture was diluted with ethyl acetate (60 mL) and water (20mL), the organic phase separated, dried over Na₂SO₄, filtered and thesolvents evaporated in vacuo. The dark residue was purified bychromatography on silica (25 g HP-SIL) using a Biotage Isolera systememploying a dichloromethane/methanol gradient (100/0→95/5→90/10→80/20)to afford the Comparative Example C7 (F-7) as off-white solid (0.033 g,63%).

¹H NMR (400 MHz, DMSO-d₆) δ=12.42 (s, 1H), 9.41 (s, 1H), 8.77 (d, 1H),8.52 (d, 1H), 8.40 (dd, 1H), 8.27 (d, 1H), 8.18 (q, 1H), 7.51 (d, 1H),7.26 (dd, 1H)

MS (ESI): m/z=265.09 [M+H]⁺

Comparative Example 7 (F-7) Precursor

Step A

To a mixture of degassed N,N′-dimethylacetamide (4 mL) in a microwavevial was added[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (0.017 g, 0.02 mmol), followed by the titlecompound from Preparative Example B (0.1 g, 0.2 mmol),bis(pinacolato)diborane (0.056 g, 0.22 mmol) and potassium acetate(0.059 g, 0.6 mmol). The reaction mixture was then heated at ˜95° C. ina sand-bath for 18 hours. The reaction mixture was diluted with ethylacetate (100 mL) and water (30 mL), the organic phase separated, driedover Na₂SO₄, filtered and the solvents evaporated in vacuo to afford thecrude title compound which was directly used in the next step.

Step B

In a 20 ml microwave tube was dissolved the crude title compound fromStep A above, 2-bromo-6-nitropyridine (0.05 g, 0.245 mmol) inN,N′-dimethylacetamide (5.10 mL). Sodium carbonate (0.408 ml, 0.816mmol) was added and the resulting stirring solution was degassed for 5minutes. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex withdichloromethane was added (0.017 g, 0.02 mmol) and the reaction mixturewas heated to 110° C. for 22 hours. TLC monitoring showed completion ofthe reaction. The reaction mixture was diluted with dichloromethane,insolubles were filtered out through Celite, and the filtrate was washedwith water three times to remove residual amounts ofN,N′-dimethylacetamide. The organic layer was dried MgSO₄, filtered andconcentrated. The residue was purified via Biotage Isolera One employingan ethyl acetate/n-heptane gradient (5/95→100/0→100/0) to afford theComparative Example C7 (F-7) Precursor as pale yellow solid (0.0174 g,16%).

¹H-NMR (400 MHz, DMSO-d₆) δ=9.43 (s, 1H), 9.38 (s, 1H), 8.81 (d, 1H),8.60 (dd, 1H), 8.33 (d, 1H), 8.28-8.24 (m, 2H), 8.18 (d, 1H), 8.10 (t,1H), 7.61 (d, 7H), 7.47 (d, 4H), 7.42 (d, 1H), 7.28 (tt, 18H), 6.58 (d,1H), 6.19 (d, 1H) MS (ESI): m/z=533.62 [M+H]⁺.

Comparative Example 8 (F-8)

Step A

To a mixture of degassed 1,4-dioxane (8 mL) in a microwave vial wasadded [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II),complex with dichloromethane (0.034 g, 0.04 mmol), followed by the titlecompound from Preparative Example B (0.2 g, 0.4 mmol),bis(pinacolato)diborane (0.112 g, 0.44 mmol) and potassium acetate(0.118 g, 1.2 mmol). The reaction mixture was then heated at ˜95° C. ina sand-bath for 18 hours. The reaction mixture was diluted with ethylacetate (100 mL) and water (30 mL), the organic phase separated, driedover Na₂SO₄, filtered and the solvents evaporated in vacuo to afford thecrude title compound which was directly used in the next step.

Step B

The crude title compound from Step A above was dissolved in a mixture ofdegassed 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave vial. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II), complexwith dichloromethane (0.034 g, 0.04 mmol), 2-bromo-5-fluoropyridine(0.086 g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol) wereadded and the reaction mixture was heated at ˜115° C. in a sand-bath for6 hours. The reaction mixture was diluted with ethyl acetate (100 mL)and water (30 mL), the organic phase separated, dried over Na₂SO₄,filtered and the solvents evaporated in vacuo. The dark residue waspurified by chromatography on silica (25 g puriFlash, Interchim) using aBiotage Isolera system employing an ethyl acetate/n-heptane gradient(5/95→100/0 →100/0) to afford a mixture of the title compound andbyproduct (0.064 g).

Step C

The mixture of the title compound and byproduct from Step B above (0.064g) was purified by preparative TLC with a loading of ˜0.03 g of mixtureper 1000 μM Analtech Uniplate (20×20 cm) using dichloromethane/acetone(90/10) as mobile phase to afford the more polar title compound asoff-white solid (0.0385 g, 18.5% for 3 steps).

¹H NMR (400 MHz, CDCl₃) δ=9.26 (s, 1H), 8.45 (d, 1H), 8.38 (AB-system,2H), 8.25 (d, 1H), 7.62-7.58 (m, 5H), 7.30-7.18 (m, 12H), 6.56 (d, 1H)

Step D

The title compound from Step C above (0.0385 g, 0.076 mmol) wasdissolved in dichloromethane (5 mL) and trifluoroacetic acid (1.2 mL)was added. The reaction mixture was stirred at room temperature for 6hours. The reaction mixture was diluted with diluted withdichloromethane (50 mL) and water (20 mL). The pH of the aqueous phasewas adjusted to pH-12 by the addition of a 1 M aqueous sodium hydroxidesolution. The aqueous layer was separated, extracted withdichloromethane (25 mL), the combined organic layer dried over Na₂SO₄,filtered and the solvent removed under reduced pressure. The residue waspurified by chromatography on silica (10 g HP-SIL-column) using aBiotage Isolera system employing a dichloromethane/methanol gradient(100/0→95/5→90/10) to afford the Comparative Example C8 (F-8) as a whitesolid (0.0079 g, 39.3%)

¹H NMR (400 MHz, DMSO-d₆) δ=12.40 (br-s, 1H), 9.40 (s, 1H), 8.77 (d,1H), 8.72 (d, 1H), 8.55-8.50 (m, 2H), 8.35 (d, 1H), 7.95-7.90 (m, 1H),7.51 (d, 1H)

MS (ESI): m/z=265.06 [M+H]⁺.

The synthesis of Comparative Example C8 (F-8) was first described inWO2016/124508 (Example 18) by a different synthesis.

Comparative Example 8 (F-8) Precursor

Step A

To a mixture of degassed 1,4-dioxane (8 mL) in a microwave vial wasadded [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II),complex with dichloromethane (0.034 g, 0.04 mmol), followed by the titlecompound from Preparative Example B (0.2 g, 0.4 mmol),bis(pinacolato)diborane (0.112 g, 0.44 mmol) and potassium acetate(0.118 g, 1.2 mmol). The reaction mixture was then heated at ˜95° C. ina sand-bath for 18 hours. The reaction mixture was diluted with ethylacetate (100 mL) and water (30 mL), the organic phase separated, driedover Na₂SO₄, filtered and the solvents evaporated in vacuo to afford thecrude title compound which was directly used in the next step.

Step B

The crude title compound from Step A above was dissolved in a mixture ofdegassed 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave vial. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II), complexwith dichloromethane (0.034 g, 0.04 mmol), 2-bromo-5-nitropyridine (0.1g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol) were added andthe reaction mixture was heated at ˜115° C. in a sand-bath for 6 hours.The reaction mixture was diluted with ethyl acetate (100 mL) and water(30 mL), the organic phase separated, dried over Na₂SO₄, filtered andthe solvents evaporated in vacuo. The dark residue was purified bychromatography on silica (25 g puriFlash, Interchim) using a BiotageIsolera system employing an ethyl acetate/n-heptane gradient (5/95→100/0→100/0) to afford a mixture of the title compound and byproduct (0.0788g).

Step C

The mixture of the title compound from Step B above and byproduct(0.0788 g) was dissolved in dichloromethane (10 mL) and trifluoroaceticacid (2.4 mL) was added. The reaction mixture was stirred at roomtemperature for 6 hours and then methanol was added (10 mL). Thesolvents were evaporated in vacuo and the residue suspended in methanol(10 mL). The solvents were again evaporated in vacuo and the residuesuspended in dichloromethane (4 mL). After the addition of triethylamine(2 mL, 14.4 mmol), di-tert-butyl dicarbonate (0.2 g, 0.86 mmol), and4-(dimethylamino)-pyridine (0.0036 g, 0.028 mmol), the reaction mixturewas stirred at room temperature for 18 hours. The reaction mixture wasdiluted with ethyl acetate (100 mL) and water (40 mL). The organic phasewas separated, dried over Na₂SO₄, filtered and the solvents removed invacuo. The residue was purified on silica (25 g puriFlash, Interchim)using a Biotage Isolera One purification system employing an ethylacetate/n-heptane gradient (5/95→100/0→100/0) to afford the ComparativeExample C8 (F-8) Precursor as pale yellow solid (0.0149 g, 25.7%).

¹H NMR (400 MHz, CDCl₃) δ=9.55 (d, 1H), 9.36 (s, 1H), 8.88 (d, 1H), 8.77(d, 1H), 8.72 (d, 1H), 8.65 (dd, 1H), 8.56 (d, 1H), 8.30 (d, 1H), 1.87(s, 9H)

MS (ESI): m/z=391.93 [M+H]⁺.

Comparative Example 9 (F-9)

Step A

To a mixture of degassed 1,4-dioxane (8 mL) in a microwave vial wasadded [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II),complex with dichloromethane (0.034 g, 0.04 mmol), followed by the titlecompound from Preparative Example B (0.2 g, 0.4 mmol),bis(pinacolato)diborane (0.112 g, 0.44 mmol) and potassium acetate(0.118 g, 1.2 mmol). The reaction mixture was then heated at ˜95° C. ina sand-bath for 18 hours. The reaction mixture was diluted with ethylacetate (100 mL) and water (30 mL), the organic phase separated, driedover Na₂SO₄, filtered and the solvents evaporated in vacuo to afford thecrude title compound which was directly used in the next step.

Step B

The crude title compound from Step A above was dissolved in a mixture ofdegassed 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave vial. Then[1,1-bis(diphenylphosphino)ferrocene]dichloro-palladium(II), complexwith dichloromethane (0.034 g, 0.04 mmol), 2-bromo-4-fluoropyridine(0.086 g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol) wereadded and the reaction mixture was heated at ˜115° C. in a sand-bath for6 hours. The reaction mixture was diluted with ethyl acetate (100 mL)and water (30 mL), the organic phase separated, dried over Na₂SO₄,filtered and the solvents evaporated in vacuo. The dark residue waspurified by chromatography on silica (25 g puriFlash, Interchim) using aBiotage Isolera system employing an ethyl acetate/n-heptane gradient(5/95→100/0 →100/0) to afford a mixture of the title compound andbyproduct (0.0489 g).

Step C

The mixture of the title compound and byproduct from Step B above(0.0489 g) was dissolved in dichloromethane (5 mL) and trifluoroaceticacid (1.5 mL) was added. The reaction mixture was stirred at roomtemperature for 6 hours. The reaction mixture was diluted with dilutedwith dichloromethane (50 mL) and water (20 mL). The pH of the aqueousphase was adjusted to pH˜12 by the addition of a 1 M aqueous sodiumhydroxide solution. The aqueous layer was separated, extracted withdichloromethane (25 mL), the combined organic layer dried over Na₂SO₄,filtered and the solvent removed under reduced pressure. The residue waspurified by preparative TLC with a loading of ˜0.03 g of mixture per1000 μM Analtech Uniplate (20×20 cm) using dichloromethane/methanol(90/10) as mobile phase to afford the less polar title compound asoff-white solid (0.0145 g, 7% for 3 steps) and a more polar mixture oftwo compounds.

¹H NMR (400 MHz, DMSO-d₆) δ=9.42 (s, 1H), 8.76 (d, 1H), 8.67 (dd, 1H),8.35 (d, 1H), 8.27 (d, 1H), 7.67-7.60 (m, 5H), 7.35-7.22 (m, 11H), 6.81(dd, 1H), 6.60 (d, 1H)

Step D

The less polar title compound from Step C above (0.0145 g, 0.027 mmol)was dissolved in dichloromethane (3 mL) and trifluoroacetic acid (2 mL)was added. The reaction mixture was stirred at room temperature for 18hours. The reaction mixture was diluted with diluted withdichloromethane (50 mL) and water (20 mL). The pH of the aqueous phasewas adjusted to pH˜12 by the addition of a 1 M aqueous sodium hydroxidesolution. The aqueous layer was separated, extracted withdichloromethane (25 mL), the combined organic layer dried over Na₂SO₄,filtered and the solvent removed under reduced pressure. The residue waspurified by chromatography on silica (10 g HP-SIL) using a BiotageIsolera system employing a dichloromethane/methanol gradient(100/0→95/5→90/10) to afford the Comparative Example C9 (F-9) asoff-white solid (0.0025 g, 33%).

¹H NMR (400 MHz, DMSO-d₆) δ=12.43 (br-s, 1H), 9.45 (s, 1H), 8.82-8.77(m, 2H), 8.54 (d, 1H), 8.44 (d, 1H), 8.22 (dd, 1H), 7.53 (d, 1H),7.46-7.42 (m, 1H)

MS (ESI): m/z=264.63 [M+H]⁺.

Comparative Example 9 (F-9) Precursor

Step A

To a mixture of degassed 1,4-dioxane (8 mL) in a microwave vial wasadded [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II),complex with dichloromethane (0.034 g, 0.04 mmol), followed by the titlecompound from Preparative Example B (0.2 g, 0.4 mmol),bis(pinacolato)diborane (0.112 g, 0.44 mmol) and potassium acetate(0.118 g, 1.2 mmol). The reaction mixture was then heated at ˜95° C. ina sand-bath for 18 hours. The reaction mixture was diluted with ethylacetate (100 mL) and water (30 mL), the organic phase separated, driedover Na₂SO₄, filtered and the solvents evaporated in vacuo to afford thecrude title compound which was directly used in the next step.

Step B

The crude title compound from Step A above was dissolved in a mixture ofdegassed 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave vial. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II), complexwith dichloromethane (0.034 g, 0.04 mmol), 2-bromo-4-nitropyridine (0.1g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol) were added andthe reaction mixture was heated at ˜115° C. in a sand-bath for 6 hours.The reaction mixture was diluted with ethyl acetate (100 mL) and water(30 mL), the organic phase separated, dried over Na₂SO₄, filtered andthe solvents evaporated in vacuo. The dark residue was purified bychromatography on silica (25 g puriFlash, Interchim) using a BiotageIsolera system employing an ethyl acetate/n-heptane gradient (5/95→100/0→100/0) to afford a mixture of the title compound and byproducts (0.076g).

Step C

The mixture of the title compound from Step B above and byproducts(0.076 g) was dissolved in dichloromethane (10 mL) and trifluoroaceticacid (2.4 mL) was added. The reaction mixture was stirred at roomtemperature for 6 hours and then methanol was added (10 mL). Thesolvents were evaporated in vacuo and the residue suspended in methanol(10 mL). The solvents were again evaporated in vacuo and the residuesuspended in dichloromethane (4 mL). After the addition of triethylamine(2 mL, 14.4 mmol), di-tert-butyl dicarbonate (0.2 g, 0.86 mmol), and4-(dimethylamino)-pyridine (0.0036 g, 0.028 mmol), the reaction mixturewas stirred at room temperature for 18 hours. The reaction mixture wasdiluted with ethyl acetate (100 mL) and water (40 mL). The organic phasewas separated, dried over Na₂SO₄, filtered and the solvents removed invacuo. The residue was purified on silica (25 g puriFlash, Interchim)using a Biotage Isolera One purification system employing an ethylacetate/n-heptane gradient (5/95→100/0→100/0) to afford the ComparativeExample C9 (F-9) Precursor and the byproduct as ˜1.1-mixture (0.0231 g,pale yellow solid).

¹H NMR (400 MHz, CDCl₃) δ=9.38 (d, 1H), 9.35 (d, 1H), 9.31 (s, 2H), 9.02(d, 1H), 8.76-8.70 (m, 5H), 8.68 (d, 1H), 8.55 (d, 1H), 8.43-8.37 (m,3H), 8.12 (dd, 1H), 8.07 (dd, 1H), 7.43 (d, 1H), 7.41 (d, 1H), 1.82 (s,18H)

MS (ESI): m/z=291.94 [MH-Boc of the title compound]⁺, 170.04 [MH⁺-Boc ofbyproduct]⁺

Comparative Example 10 (F-10)

Step A

To a mixture of degassed 1,4-dioxane (8 mL) in a microwave vial wasadded [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II),complex with dichloromethane (0.034 g, 0.04 mmol), followed by the titlecompound from Preparative Example B (0.2 g, 0.4 mmol),bis(pinacolato)diborane (0.112 g, 0.44 mmol) and potassium acetate(0.118 g, 1.2 mmol). The reaction mixture was then heated at ˜95° C. ina sand-bath for 18 hours. The reaction mixture was diluted with ethylacetate (100 mL) and water (30 mL), the organic phase separated, driedover Na₂SO₄, filtered and the solvents evaporated in vacuo to afford thecrude title compound which was directly used in the next step.

Step B

The crude title compound from Step A above was dissolved in a mixture ofdegassed 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave vial. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II), complexwith dichloromethane (0.034 g, 0.04 mmol), 2-bromo-3-fluoropyridine(0.086 g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol) wereadded and the reaction mixture was heated at ˜115° C. in a sand-bath for6 hours. The reaction mixture was diluted with ethyl acetate (100 mL)and water (30 mL), the organic phase separated, dried over Na₂SO₄,filtered and the solvents evaporated in vacuo. The dark residue waspurified by chromatography on silica (25 g puriFlash, Interchim) using aBiotage Isolera system employing an ethyl acetate/n-heptane gradient(5/95→100/0 →100/0) to afford a mixture of the title compound andbyproduct (0.0586 g).

Step C

The mixture of the title compound and byproduct from Step B above(0.0586 g) was dissolved in dichloromethane (5 mL) and trifluoroaceticacid (1.8 mL) was added. The reaction mixture was stirred at roomtemperature for 6 hours. The reaction mixture was diluted with dilutedwith dichloromethane (50 mL) and water (20 mL). The pH of the aqueousphase was adjusted to pH-12 by the addition of a 1 M aqueous sodiumhydroxide solution. The aqueous layer was separated, extracted withdichloromethane (25 mL), the combined organic layer dried over Na₂SO₄,filtered and the solvent removed under reduced pressure. The residue waspurified by chromatography on silica (10 g HP-SIL) using a BiotageIsolera system employing a dichloromethane/methanol gradient(100/0→95/5→90/10) to afford the Comparative Example C10 (F-10) asoff-white solid (0.0067 g, 5.7% for 3 steps).

¹H NMR (400 MHz, DMSO-d₆) δ=12.47 (br-s, 1H), 9.45 (s, 1H), 8.80 (d,1H), 8.63-8.61 (m, 1H), 8.55-8.53 (m, 1H), 8.00 (d, 1H), 7.94-7.88 (m,1H), 7.63-7.58 (m, 1H), 7.52 (d, 1H)

MS (ESI): m/z=264.84 [M+H]⁺.

Comparative Example 10 (F-10) Precursor

Step A

To a mixture of degassed 1,4-dioxane (8 mL) in a microwave vial wasadded [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II),complex with dichloromethane (0.034 g, 0.04 mmol), followed by the titlecompound from Preparative Example B (0.2 g, 0.4 mmol),bis(pinacolato)diborane (0.112 g, 0.44 mmol) and potassium acetate(0.118 g, 1.2 mmol). The reaction mixture was then heated at ˜95° C. ina sand-bath for 18 hours. The reaction mixture was diluted with ethylacetate (100 mL) and water (30 mL), the organic phase separated, driedover Na₂SO₄, filtered and the solvents evaporated in vacuo to afford thecrude title compound which was directly used in the next step.

Step B

The crude title compound from Step A above was dissolved in a mixture ofdegassed 1,4-dioxane (8.6 mL) and water (2 mL) in a microwave vial. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II), complexwith dichloromethane (0.034 g, 0.04 mmol), 2-bromo-3-nitropyridine (0.1g, 0.49 mmol) and cesium carbonate (0.266 g, 0.82 mmol) were added andthe reaction mixture was heated at ˜115° C. in a sand-bath for 6 hours.The reaction mixture was diluted with ethyl acetate (100 mL) and water(30 mL), the organic phase separated, dried over Na₂SO₄, filtered andthe solvents evaporated in vacuo. The dark residue was purified bychromatography on silica (25 g puriFlash, Interchim) using a BiotageIsolera system employing an ethyl acetate/n-heptane gradient (5/95→100/0→100/0) to afford a mixture of the title compound and byproduct (0.0538g).

Step C

The mixture of the title compound from Step B above and byproduct(0.0538 g) was dissolved in dichloromethane (4 mL) and trifluoroaceticacid (2.5 mL) was added. The reaction mixture was stirred at roomtemperature for 16 hours and then methanol was added (10 mL). Thesolvents were evaporated in vacuo and the residue suspended in methanol(10 mL). The solvents were again evaporated in vacuo and the residuesuspended in dichloromethane (4 mL). After the addition of triethylamine(2 mL, 14.4 mmol), di-tert-butyl dicarbonate (0.2 g, 0.86 mmol), and4-(dimethylamino)-pyridine (0.0036 g, 0.028 mmol), the reaction mixturewas stirred at room temperature for 18 hours. The reaction mixture wasdiluted with ethyl acetate (100 mL) and water (40 mL). The organic phasewas separated, dried over Na₂SO₄, filtered and the solvents removed invacuo. The residue was purified on silica (25 g puriFlash, Interchim)using a Biotage Isolera One purification system employing an ethylacetate/n-heptane gradient (5/95→100/0→100/0) to afford the ComparativeExample C10 (F-10) Precursor as pale yellow solid (0.0194 g, 12.1% for 3steps).

¹H NMR (400 MHz, CDCl₃) δ=9.35 (d, 1H), 8.90 (d, 1H), 8.73 (d, 1H), 8.58(d, 1H), 8.24-8.17 (m, 3H), 7.57-7.53 (m, 1H), 1.73 (s, 9H)

MS (ESI): m/z=391.92 [MH⁺], 291.90 [MH⁺-Boc]

Synthesis of ¹⁸F-Labeled Compounds

General ¹⁸F-Fluorination Method a (Direct Aromatic ¹⁸F-Fluorination)

The n.c.a [¹⁸F]fluoride (2-5 GBq) was trapped on a Sep-Pak Accell PlusQMA light cartridge (Waters) and eluted with a solution K₂CO₃/Kryptofix®2.2.2. The water was removed using a stream of N₂ at 120° C. andco-evaporated to dryness with MeCN (3×1 mL). Afterwards, a solution ofthe dissolved precursor was added to the dried K[¹⁸F]F-K₂₂₂ complex. Thereaction vial was sealed and heated under conventional heating for 15min at 130° C. Subsequently, the reaction mixture was quenched withwater and the crude product was purified via semi-preparative HPLC. Theisolated tracer was diluted with water (35 mL), trapped on a C-18 Pluscartridge (Waters), washed with water (5 mL), eluted with ethanol (1 mL)and formulated in saline.

General ¹⁸F-Fluorination Method (Direct ¹⁸F-Labeling Plus Deprotection)

The tracers were synthesized starting from n.c.a. [¹⁸F]fluoride (1-10GBq) by a ¹⁸F-direct fluorination. The aqueous [¹⁸F]fluoride solutionwas trapped on a Sep-Pak Accell Plus QMA light cartridge (Waters) andeluted with a solution K₂CO₃/Kryptofix® 2.2.2. The water was removedusing a stream of N₂ at 120° C. and co-evaporated to dryness with MeCN(3×1 mL). Afterwards, the respective dissolved precursor was added tothe dried K[¹⁸F]F-K₂₂₂ complex. The reaction vial was sealed and heatedfor 15 min at 120-160° C. (heating block).

For deprotection hydrochloric acid was added and the mixture was stirredfor another 10 min at 110° C. After neutralization using sodiumhydroxide solution the reaction mixture was quenched with ammoniumformate buffer and trapped on a C-18 Plus cartridge (Waters). Thecartridge was washed with water (5 mL), eluted with acetonitrile andsubsequently, the crude product was purified via semi-preparative HPLC.The isolated tracer was diluted with water (25 mL), trapped on a C-18Plus cartridge (Waters), washed with water (5 mL), eluted with ethanol(1 mL) and formulated in saline.

Example ¹⁸F-1

¹⁸F-1 (680 MBq) was synthesized according to General ¹⁸F-fluorinationmethod A using the corresponding nitro precursor molecule (M. Timothy etal., J. Labelled Comp. Radiopharm. (2013), 56(14), 736-740) (2.8 mg, 7.1μmol) in dimethyl sulfoxide (0.6 mL).

The radiochemical purity of 100% was determined by analyticalreversed-phase HPLC (t_(R)(RAD-trace)=3.19 min). The identity of ¹⁸F-1was confirmed by comparing the retention time with the non-radioactivereference F-1.

Example ¹⁸F-2

¹⁸F-2 (680 MBq) was synthesized according to General ¹⁸F-fluorinationmethod A using Comparative Example 2 (F-2) precursor (WO 2015/052105)(3.4 mg, 8.7 μmol) in dimethyl sulfoxide (0.6 mL).

The radiochemical purity of 98% was determined by analyticalreversed-phase HPLC (t_(R)(RAD-trace)=3.27 min). The identity of ¹⁸F-2was confirmed by comparing the retention time with the non-radioactivereference F-2.

Example ¹⁸F-4

¹⁸F-4 (300 MBq) was synthesized according to General ¹⁸F-fluorinationmethod B using precursor molecule compound 14 (3.1 mg, 5.9 μmol) indimethyl sulfoxide (0.6 mL).

The radiochemical purity of 100% was determined by analyticalreversed-phase HPLC (t_(R)(RAD-trace)=3.31 min). The identity of ¹⁸F-4was confirmed by comparing the retention time with the non-radioactivereference F-4.

Determination of Binding in AD and Healthy Control Brain Homogenates

20 μg of human Alzheimer disease brain homogenate was incubated with adilution series of each test compound (1000 to 0.06 nM) in the presenceof 800 Bq of ¹⁸F-labeled Tau binder. The samples were shaken at 110 rpmfor 45 min at 37° C. Samples were then filtered through GF/B 96 wellfilter plates and washed twice with 300 μL assay buffer (PBS containing0.1% BSA and 2% DMSO). Thereafter, filter plates were sealed and a FujiFilm Imaging Plate (BAS-SR2025) was placed on top. The imaging plate wasanalyzed after overnight exposition using a Fuji Film BAS-5000.Non-specific signal was determined with samples containing ¹⁸F-labeledTau-reference binder in the presence of assay buffer without brainsubstrate and competitor. Specific binding was calculated by subtractingthe non-specific signal from the measured samples signal. The unblocked¹⁸F-labeled Tau-binder signal was defined as total binding. IC₅₀ valueswere calculated by Prism V6 (GraphPad) setting total binding to 100%.

Results:

High tau-affinity of compounds F-1, F-2, and F-4 was found in acompetition assay using human AD brain homogenate. IC₅₀ values for Taubinding of <2 nM were measured for all compounds.

A high signal-to-noise ratio between AD brain homogenate and healthycontrol brain homogenate was obtained with the compound ¹⁸F-4 with aratio of 10.1. A low signal to noise ratio between AD brain homogenateand healthy control brain homogenate of 1.3 was obtained for compound¹⁸F-1.

Further data were generated using additional human brain tissues.

The signal-to-noise ratio between AD brain homogenate and healthycontrol brain homogenate for compound ¹⁸F-4 was determined to be 12.2,21.0, 27.5 respectively.

Significant lower values were obtained for compound ¹⁸F-1, where thoseratios are only 1.7, 1.8 and 2.5.

Also compound ¹⁸F-2 showed significantly lower ratios between the signalin AD brain homogenates and healthy control brain homogenate (3.3, 4.5,6.9).

Autoradiography in Human Brain Slices

18 micron thick frozen human brain slices and 6 micron thick human FFPEbrain slices were examined via autoradiography. Brain sections wereequilibrated for at least 1 h in 1×PBS solution prior to use in theexperiment. Each brain section was covered with a solution of the¹⁸F-labeled tracer (200 Bq/μl, 500 μl) in 1×PBS. For blockingexperiments with the corresponding ¹⁹F-compound, an excess of theblocking compound (10 μM) was mixed with the ¹⁸F-compound. The brainsections were allowed to incubate with the tracer solution at roomtemperature for 1 h, drained afterwards and placed in a slide holder.The slides were then washed sequentially with 1×PBS for 1 min; 70% EtOHin 1×PBS for 2 min; 30% EtOH in 1×PBS for 2 min; and 1×PBS for 1 min.The slides were allowed to air-dry before being placed on Fuji imagingplates for 30 min for overnight exposure. The imaging plates werescanned and the signal was measured using Fuji software to produce anautoradiographic image of the brain section.

Results:

Compound ¹⁸F-4 was tested in autoradiography studies using human brainsections (AD, PSP, PiD, HC). Using section from AD brains, a strongpunctated staining was detectable that could be blocked with theaddition of excess corresponding cold compound. In the healthy control(HC) sections, no specific signal was visible (FIG. 1). Similar resultswere obtained for compound ¹⁸F-4 on PSP and PiD brain sections.

Determination of the Binding Affinity to Amyloid-Beta in AD BrainHomogenate

20 μg of human Alzheimer disease brain homogenate was incubated with adilution series of each test compound (1000 to 0.06 nM) in the presenceof 800 Bq of ¹⁸F-labeled beta-amyloid binder. The samples were shaken at110 rpm for 45 min at 37° C. Samples were then filtered through GF/B 96well filter plates and washed twice with 300 μL assay buffer (PBScontaining 0.1% BSA and 2% DMSO). Thereafter, filter plates were sealedand a Fuji Film Imaging Plate (BAS-SR2025) was placed on top. Theimaging plate was analyzed after overnight exposition using a Fuji FilmBAS-5000. Non-specific signal was determined with samples containing¹⁸F-labeled beta-amyloid binder in the presence of assay buffer withoutbrain substrate and competitor. Specific binding was calculated bysubtracting the non-specific signal from the measured samples signal.The unblocked ¹⁸F-labeled beta-amyloid binder signal was defined astotal binding. IC₅₀ values were calculated by Prism V6 (GraphPad)setting total binding to 100%.

Results:

Low affinity of compounds F-1, F-2, and F-4 for beta-amyloid was foundin a competition assay using human AD brain homogenate. IC₅₀ values forbeta-amyloid binding of >1 μM were measured for all compounds.

Determination of the Binding Affinity to MAO A in HC Brain Homogenate

20 μg of human brain homogenate (without AD pathology) was incubatedwith a dilution series of each test compound (1000 to 0.06 nM) in thepresence of 800 Bq of ¹⁸F-labeled MAO-A binder ([¹⁸F]fluoroethylharmine, FEH). The samples were shaken at 110 rpm for 45 min at 37° C.Samples were then filtered through GF/B 96 well filter plates and washedtwice with 300 μL assay buffer (PBS containing 0.1% BSA and 2% DMSO).Thereafter, filter plates were sealed and a Fuji Film Imaging Plate(BAS-SR2025) was placed on top. The imaging plate was analyzed afterovernight exposition using a Fuji Film BAS-5000. Non-specific signal wasdetermined with samples containing ¹⁸F-labeled FEH in the presence ofassay buffer without brain substrate and competitor. Specific bindingwas calculated by subtracting the non-specific signal from the measuredsamples signal. The unblocked ¹⁸F-labeled FEH signal was defined astotal binding. IC₅₀ values were calculated by Prism V6 (GraphPad)setting total binding to 100%.

Results:

In the mouse brain homogenate, compound F-1 showed a high off-targetaffinity towards MAO A of 22 nM in the ¹⁸F-FEH competition assay. Theaffinity of compound F-2 was reduced to 475 nM, whereas off-targetaffinity to MAO A for compound F-4 was further reduced with IC₅₀ valuesof 1300 nM.

Using human control brain homogenate (healthy control) compound F-1showed a high off-target affinity towards MAO A of 5 nM in the FEHcompetition assay. The affinity of compound F-2 was reduced to 100 nM,whereas off-target affinity to MAO A for compound F-4 was furtherreduced with an IC₅₀ value of 530 nM.

Determination of the Binding Affinity to MAO B in HC Brain Homogenate

20 μg of human brain homogenate (without AD pathology) was incubatedwith a dilution series of each test compound (1000 to 0.06 nM) in thepresence of 800 Bq of ¹⁸F-labeled MAO-B binder ([¹⁸F]fluoro deprenyl).The samples were shaken at 110 rpm for 45 min at 37° C. Samples werethen filtered through GF/B 96 well filter plates and washed twice with300 μL assay buffer (PBS containing 0.1% BSA and 2% DMSO). Thereafter,filter plates were sealed and a Fuji Film Imaging Plate (BAS-SR2025) wasplaced on top. The imaging plate was analyzed after overnight expositionusing a Fuji Film BAS-5000. Non-specific signal was determined withsamples containing ¹⁸F-labeled fluoro deprenyl in the presence of assaybuffer without brain substrate and competitor. Specific binding wascalculated by subtracting the non-specific signal from the measuredsamples signal. The unblocked ¹⁸F-labeled fluoro deprenyl signal wasdefined as total binding. IC₅₀ values were calculated by Prism V6(GraphPad) setting total binding to 100%.

Results:

In the human HC brain homogenate, compound F-1 showed a high off-targetaffinity towards MAO B of 170 nM in the ¹⁸F-labeled fluoro deprenylcompetition assay. The affinity of compound F-4 was reduced tovalues >1000 nM.

PK Studies in Healthy Mice

NMRI mice (weight range 25-35 g) were injected intravenously with the¹⁸F-labeled compounds. Up to 150 μL of 1×PBS solution with 10%-15% EtOHor dilution medium (57% water for injections, 18% polyethylene glycol400, 15% ethanol, 10% water) containing the ¹⁸F-labeled compound (2-10MBq) were injected. Anesthesia with isoflurane was induced beforeinjection of the tracer and maintained during the image acquisitionperiod. PET scans were performed using a SIEMENS INVEON small animalPET/CT scanner (Siemens, Knoxville, Tenn.). PET acquisition was startedimmediately before the radioactive dose was injected into the animalthrough the tail vein. Images were generated as dynamic scans for 60minutes.

Compound ¹⁸F-1: peak uptake: 5.3% ID/g, ratio of uptake peak/30 min:6.8, brain retention at 60 min: 0.8% ID/g, bone uptake in shoulder jointat 60 min: 4.0% ID/g.

Compound ¹⁸F-2: peak uptake: 5.7% ID/g, ratio of uptake peak/30 min:10.9, brain retention at 60 min: 0.6% ID/g, bone uptake in shoulderjoint at 60 min: 6.2% ID/g.

Compound ¹⁸F-4: peak uptake: 5.8% ID/g, ratio of uptake peak/30 min:22.0, brain retention at 60 min: 0.2% ID/g, bone uptake in shoulderjoint at 60 min: not detectable.

The peak uptake in the brain was set to 100% and washout curves weregenerated to evaluate the clearance of the activity from the normalbrain (FIG. 2).

Human Imaging Study

In a clinical trial, subjects with AD as well as non-demented controls(NDC) underwent dynamic PET imaging for over 3 h following 370 MBq bolusinjection of ¹⁸F-4.

Results:

Initial imaging data shows robust brain uptake and fast wash-out innon-target regions. In NDCs, there was no increased uptake seen inchoroid plexus, basal ganglia, striatum, amygdala, meninges or otherregions noted with other tau agents (FIG. 4a ). ¹⁸F-4 shows good brainuptake and fast washout from non-target brain regions (see FIG. 3). InAD, focal asymmetric uptake was evident in temporal, parietal andfrontal lobes (FIG. 4b ).

TABLE 1 Summary of pre-clinical characteristics Criteria forTau-PET-Imaging agents Compound ¹⁸F-1 Compound ¹⁸F-2 Compound ¹⁸F-4 Highaffinity to Tau +++  +++ +++ (IC₅₀ in AD brain homogenate)^(b)) (<2nM)^(a)) (<2 nM)^(a)) (<2 nM)^(a)) Binding to AD and non-AD tauopathies(determined by autoradiography on human brain slices)^(c)) AD brain(Braak stage 5/6) +++^(d))  +++^(e))  +++^(a)) AD brain (Braak stage1/3) NA NA  +++^(a)) PSP  −^(d)) −^(e))  +++^(a)) PiD  −^(d)) −^(e)) +++^(a)) Low affinity to amyloid-beta +++^(a))  +++^(a))  +++^(a))(IC₅₀ in AD brain homogenate)^(b)) >1 μM >1 μM >1 μM Low affinity to MAOA − + +++ (IC₅₀ in mouse brain homogenate)^(b)) (22 nM)^(a)) (475nM)^(a)) (1300 nM)^(a)) Low affinity to MAO A − ∘ ++ (IC₅₀ in HChomogenate)^(b)) (5 nM)^(a)) (100 nM)^(a)) (530 nM)^(a)) Low affinity toMAO B ∘ +++ +++ (IC₅₀ in HC homogenate)^(b)) (170 nM) (>1000 nM) (>1000nM) High signal to noise − +++ (Ratio of tracer signal AD brain/signal1.3^(a)) NA 10.1^(a)) in HC brain homogenate)^(c)) High signal to noise(further − ∘ +++ homogenates) Ratio of tracer signal in 3 different AD1.7, 1.8, 2.5^(a)) 3.3, 4.5, 6.9^(a)) 12.2, 21.0, 27.5^(a)) brainhomogenates/signal in HC brain homogenates)^(c)) High signal to noise ∘+++ +++ (Ratio of tracer signal AD brain/signal 2.2^(a)) 15.0^(a))15.1^(a)) in mouse brain homogenate)^(c)) Good brain uptake ++ ++ ++(Tracer uptake in healthy mice after iv (5.3% ID/g)^(a)) (5.7%ID/g)^(a)) (5.8% ID/g)^(a)) injection)^(c)) Fast washout from healthybrain ∘ + +++ (Ratio of tracer uptake at 2 min and 30 (6.8)^(a))(10.9)^(a)) (22.0)^(a)) min in healthy mice)^(c)) Low retention inhealthy brain − ∘ +++ (Tracer signal in healthy mice at 60 min (0.8%ID/g)^(a)) (0.6% ID/g)^(a)) (0.2% ID/g)^(a)) after iv injection)^(c))Low or minor de-fluorination ∘ −  +++ (Tracer uptake in bone in healthymice (4.0% ID/g)^(a)) (6.2% ID/g)^(a)) (<0.5% ID/g)^(a), f)) at 60 minafter iv injection)^(c)) − poor, ∘ moderate, + good, ++ very good, +++excellent ^(a))in house data, see experimental section above;^(b))determined with the non-radioactive fluorine-19 derivatives F-1,F-2 and F-4; ^(c))determined with the radioactive fluorine-18derivatives ¹⁸F-1, ¹⁸F-2 and ¹⁸F-4; ^(d))Marquie et al. 2015;^(e))WO2015/052105; ^(f))no de-fluorination detected NA: not available.

As can be seen from Table 1, the prior art compounds ¹⁸F-1 and ¹⁸F-2have limitations especially in respect to:

-   -   Low binding to Tau-isoforms in non-AD tauopathies,    -   Affinity for MAO A, and thus low selectivity to Tau,    -   Not having low signal in healthy brain,    -   Not having fast washout from healthy brain,    -   Long-term retention in healthy brain, and/or    -   in vivo de-fluorination.

On the other hand, compound ¹⁸F-4 shows:

-   -   Specific binding to AD and non-AD tauopathy brain slices        (examples: strong signal for

PSP, PiD in contrast to the reports for compound ¹⁸F-1),

-   -   Less affinity to MAO A in whole mouse brain homogenate (59-fold        higher IC₅₀ than compound ¹⁸F-1 and 2.7-fold higher IC₅₀ than        compound ¹⁸F-2),    -   Less affinity to MAO A in HC brain homogenate (106-fold higher        IC₅₀ than compound ¹⁸F-1 and 5-fold higher IC₅₀ than compound        ¹⁸F-2),    -   Less affinity to MAO B in HC brain homogenate (>5-fold higher        IC₅₀ than compound ¹⁸F-1),    -   Higher signal to noise ratio, determined by the binding in AD        brain homogenate vs. HC brain homogenate (7.7-fold higher ratio        than ¹⁸F-1),    -   Higher signal to noise ratio, determined by the binding in        further AD brain homogenate vs. HC brain homogenate (7.2-11 fold        higher than ¹⁸F-1, 3.7-4.0 fold higher ratio than ¹⁸F-2),    -   Higher signal to noise ratio, determined by the binding in AD        brain homogenate vs. whole mouse brain homogenate (6.9-fold        higher ratio than ¹⁸F-1),    -   Faster washout from healthy brain in mice (3.2-fold faster than        compound ¹⁸F-1 and 2.0-fold faster than compound ¹⁸F-2),    -   Lower long-term retention in healthy brain (4-fold less than        compound ¹⁸F-1 and 3-fold less than compound ¹⁸F-2),    -   No de-fluorination in mice (no bone uptake in contrast to 4.0%        ID/g for compound ¹⁸F-1 and 6.2% ID/g for compound ¹⁸F-2),

Due at least to its high affinity to Tau, its faster brain-washout,lower long-term retention in healthy brain, and/or lower bindingaffinity to other brain targets, compound ¹⁸F-4 has significantly betterproperties for determining and quantifying Tau deposits in the brain bypositron emission tomography than the prior art compounds ¹⁸F-1 and¹⁸F-2. In addition to the detection and quantification of Tau depositsin AD, compound ¹⁸F-4 can be useful for clinical evaluation of non-ADtauopathies.

The favorable pre-clinical characteristics of ¹⁸F-4 have been confirmedin human subjects. ¹⁸F-4 shows good brain uptake and fast washout fromnon-target brain regions (see FIG. 3).

The uptake pattern observed in AD was in accordance with the expectedpattern of Tau pathology (FIG. 4).

Surprisingly, ¹⁸F-4 shows significant advantages compared to itsregioisomers regarding the key features of a Tau PET imaging tracer(Table 2).

The binding affinity for Tau was poor for the comparative examples6/¹⁸F-6 and 10/¹⁸F-10 and inferior for the comparative examples 5/¹⁸F-5,7/¹⁸F-7 and 9/¹⁸F-9 (IC₅₀ determined in AD brain homogenate).

Inferior selectivity over MAO A was found for the comparative examples2/¹⁸F-2, 7/¹⁸F-7, and 8/¹⁸F.

The radiolabeling of the comparative examples ¹⁸F-5, ¹⁸F-6, ¹⁸F-8,¹⁸F-9, and ¹⁸F-10 was inferior (or failed) using standard conditions.

Poor brain uptake in mice was found for comparative example ¹⁸F-10.

The washout in healthy mouse brain was inferior for comparative examples¹⁸F-5, ¹⁸F-7 and ¹⁸F-10.

De-fluorination in mice was found for the comparative examples ¹⁸F-2,¹⁸F-5, ¹⁸F-7 and ¹⁸F-10.

TABLE 2 Comparison of regio isomers high low Tau MAO A Mouse PK bindingbinding Radio- Wash- Example [nM]^(a,b) [nM]^(a,b,d) labeling^(a)Uptake^(a) out^(a) De-fluorination^(a) 4/¹⁸F-4 +++ +++ +++ ++ ++ +++ (<5 nM) (>1000 nM) (5.8% ID/g) (22)   (<0.5% ID/g) ^(f) Comparative+++ + +++ ++ ++ − example 2/¹⁸F-2  (<5 nM)  (475 nM) (5.7% ID/g) (10.9) (6.2% ID/g) Comparative ++ +++ + ++ + + example 5/¹⁸F-5 (6.3 nM) (>1000nM) (5.3% ID/g) (9.6) (2.9% ID/g) Comparative − +++ ∘ +++ ++ ∘ example6/¹⁸F-6  (43 nM) (>1000 nM) (8.2% ID/g) (14.5)  (4.8% ID/g) Comparative++ + + +++ ∘ − example 7/¹⁸F-7 (7.1 nM)   (268 nM)) (8.5% ID/g) (7.0)(11.1% ID/g)  Comparative +++ ∘ − NA NA NA example 8/¹⁸F-8  (<5 nM)   (75 nM) Comparative ++ +++ − NA NA NA example 9/¹⁸F-9 (8.4 nM) (>1000nM) Comparative − +++ ∘ ∘ − − example 10/¹⁸F-10 (103 nM)  (>1000 nM)(2.1% ID/g) (3.6) (6.2% ID/g) − poor, ∘ moderate, + good, ++ very good,+++ excellent ^(a)in house data, see experimental section above;^(b)determined with the corresponding non-radioactive fluorine-19derivatives c) determined with the radioactive fluorine-18 derivatives¹⁸F-1, ¹⁸F-2 and ¹⁸F-4; ^(d)IC₅₀ in mouse brain homogenate ^(f) node-fluorination detected NA: not available.

The invention claimed is:
 1. A compound of the formula (I)

as well as pharmaceutically acceptable salts, hydrates, solvates,prodrugs and polymorphs thereof; wherein R¹ is ¹⁸F and R² is H.
 2. Acompound of the formula (I)

as well as pharmaceutically acceptable salts, hydrates, solvates,prodrugs and polymorphs thereof; wherein R¹ is F and R² is H.
 3. Acompound of the formula (I)

as well as pharmaceutically acceptable salts, hydrates, solvates,prodrugs and polymorphs thereof; R¹ is LG, wherein LG is nitro, halogenor trimethyl ammonium; R² is H or PG; PG is a protecting group; LG is aleaving group.
 4. The compound according to claim 3, wherein PG istert-butyloxycarbonyl (BOC), triphenylmethyl (Trityl) or dimethoxytrityl(DMT).
 5. The compound according to claim 2, wherein the compound isdetectably labeled with ²H, ³H.
 6. A diagnostic composition comprising acompound as defined in claim 1 and optionally a pharmaceuticallyacceptable carrier, diluent, adjuvant or excipient.
 7. A method ofpreparing a compound as defined in claim 1 comprising reacting acompound of the formula (I)

wherein R¹ is a leaving group (LG) and R² is H or protecting group (PG),with a [¹⁸F]fluorinating agent, wherein the method further comprisescleaving of the protecting group PG, if present.
 8. A kit for preparinga radiopharmaceutical preparation, said kit comprising a sealed vialcontaining a predetermined quantity of a compound as defined in claim 3.9. A method of collecting data for the diagnosis of a disorderassociated with tau aggregates in a sample or a patient comprising: (a)bringing a sample or a specific body part or body area suspected tocontain a tau aggregate into contact with a compound as defined in claim5; (b) allowing the compound to bind to the tau aggregate; (c) detectingthe compound bound to the tau aggregate; and (d) collecting data for thediagnosis of the disorder associated with tau aggregates in the sampleor the specific body part or body area; (e) optionally correlating thepresence or absence of compound binding with the tau aggregate with thepresence or absence of tau aggregate in the sample or specific body partor body area.
 10. A method of determining the amount of tau aggregate ina tissue and/or a body fluid comprising: (a) providing a samplerepresentative of the tissue and/or body fluid under investigation; (b)bringing the sample into contact with a compound as defined in claim 5;(c) testing the sample for the presence of tau aggregate with thecompound; (d) determining the amount of compound bound to the tauaggregate; and (e) calculating the amount of tau aggregate in the tissueand/or body fluid.
 11. A method of collecting data for determining apredisposition to a disorder associated with tau aggregates in a patientcomprising detecting the specific binding of a compound as defined inclaim 5 to a tau aggregate in a sample or in situ which comprises thesteps of: (a) bringing the sample or a specific body part or body areasuspected to contain the tau aggregate into contact with the compound asdefined in claim 5, which compound specifically binds to the tauaggregate; (b) allowing the compound to bind to the tau aggregate toform a compound/tau aggregate complex; (c) detecting the formation ofthe compound/tau aggregate complex; (d) collecting data for determiningthe predisposition to the disorder associated with tau aggregates in thepatient; (e) optionally correlating the presence or absence of thecompound/tau aggregate complex with the presence or absence of tauaggregate in the sample or specific body part or body area; and (f)optionally comparing the amount of the compound/tau aggregate to anormal control value.
 12. A method of collecting data for monitoringresidual disorder in a patient suffering from a disorder associated withtau aggregates who has been treated with a medicament, wherein themethod comprises: (a) bringing a sample or a specific body part or bodyarea suspected to contain a tau aggregate into contact with a compoundas defined in claim 5, which compound specifically binds to the tauaggregate; (b) allowing the compound to bind to the tau aggregate toform a compound/tau aggregate complex; (c) detecting the formation ofthe compound/tau aggregate complex; (d) collecting data for monitoringresidual disorder in the patient suffering from the disorder associatedwith tau aggregates who has been treated with the medicament; (e)optionally correlating the presence or absence of the compound/tauaggregate complex with the presence or absence of tau aggregate in thesample or specific body part or body area; and (f) optionally comparingthe amount of the compound/tau aggregate to a normal control value. 13.A method of collecting data for predicting responsiveness of a patientsuffering from a disorder associated with tau aggregates and beingtreated with a medicament comprising: (a) bringing a sample or aspecific body part or body area suspected to contain an tau aggregateinto contact with a compound as defined in claim 5, which compoundspecifically binds to the tau aggregate; (b) allowing the compound tobind to the tau aggregate to form a compound/tau aggregate complex; (c)detecting the formation of the compound/tau aggregate complex; (d)collecting data for predicting responsiveness of the patient sufferingfrom the disorder associated with tau aggregates and being treated withthe medicament; (e) optionally correlating the presence or absence ofthe compound/tau aggregate complex with the presence or absence of tauaggregate in the sample or specific body part or body area; and (f)optionally comparing the amount of the compound/tau aggregate to anormal control value.
 14. The method of claim 9, wherein the disorderassociated with tau aggregates is a tauopathy, Alzheimer's disease (AD),familial AD, Creutzfeldt-Jacob disease, dementia pugilistica, Down'sSyndrome, Gerstmann-Sträussler-Scheinker disease, inclusion-bodymyositis, prion protein cerebral amyloid angiopathy, traumatic braininjury, amyotrophic lateral sclerosis, Parkinsonism-dementia complex ofGuam, non-Guamanian motor neuron disease with neurofibrillary tangles,argyrophilic grain disease, corticobasal degeneration, diffuseneurofibrillary tangles with calcification, frontotemporal dementia withParkinsonism linked to chromosome 17, Hallervorden-Spatz disease,multiple system atrophy, Niemann-Pick disease type C,pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy (PSP), subacutesclerosing panencephalitis, tangle only dementia, postencephaliticParkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like withastrocytes, certain prion diseases (GSS with Tau), mutations in LRRK2,chronic traumatic encephalopathy, familial British dementia, familialDanish dementia, frontotemporal lobar degeneration, GuadeloupeanParkinsonism, neurodegeneration with brain iron accumulation,SLC9A6-related mental retardation, white matter tauopathy with globularglial inclusions, traumatic stress syndrome, epilepsy, Lewy bodydementia (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutchtype), mild cognitive impairment (MCI), multiple sclerosis, Parkinson'sdisease, HIV-related dementia, adult onset diabetes, senile cardiacamyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primaryretinal degeneration, macular degeneration, optic nerve drusen, opticneuropathy, optic neuritis, and lattice dystrophy, or atypicalparkinsonism.
 15. The method of claim 14, wherein the tauopathy is a 3Rtauopathy or a 4R tauopathy.
 16. The method of claim 9, wherein the bodypart or body area comprises the brain or the eye.
 17. The method ofclaim 9, wherein the detecting comprises positron emission tomographyimaging.
 18. A method of collecting data for the diagnosis of a disorderassociated with tau aggregates in a sample or a patient comprising: (a)bringing a sample or a specific body part or body area suspected tocontain a tau aggregate into contact with a compound as defined in claim1; (b) allowing the compound to bind to the tau aggregate; (c) detectingthe compound bound to the tau aggregate; and (d) collecting data for thediagnosis of the disorder associated with tau aggregates in the sampleor the specific body part or body area; (e) optionally correlating thepresence or absence of compound binding with the tau aggregate with thepresence or absence of tau aggregate in the sample or specific body partor body area.
 19. A method of determining the amount of tau aggregate ina tissue and/or a body fluid comprising: (a) providing a samplerepresentative of the tissue and/or body fluid under investigation; (b)bringing the sample into contact with a compound as defined in claim 1;(c) testing the sample for the presence of tau aggregate with thecompound; (d) determining the amount of compound bound to the tauaggregate; and (e) calculating the amount of tau aggregate in the tissueand/or body fluid.
 20. A method of collecting data for determining apredisposition to a disorder associated with tau aggregates in a patientcomprising detecting the specific binding of a compound as defined inclaim 1 to a tau aggregate in a sample or in situ which comprises thesteps of: (a) bringing the sample or a specific body part or body areasuspected to contain the tau aggregate into contact with the compound asdefined in claim 1, which compound specifically binds to the tauaggregate; (b) allowing the compound to bind to the tau aggregate toform a compound/tau aggregate complex; (c) detecting the formation ofthe compound/tau aggregate complex; (d) collecting data for determiningthe predisposition to the disorder associated with tau aggregates in thepatient; (e) optionally correlating the presence or absence of thecompound/tau aggregate complex with the presence or absence of tauaggregate in the sample or specific body part or body area; and (f)optionally comparing the amount of the compound/tau aggregate to anormal control value.
 21. A method of collecting data for monitoringresidual disorder in a patient suffering from a disorder associated withtau aggregates who has been treated with a medicament, wherein themethod comprises: (a) bringing a sample or a specific body part or bodyarea suspected to contain a tau aggregate into contact with a compoundas defined in claim 1, which compound specifically binds to the tauaggregate; (b) allowing the compound to bind to the tau aggregate toform a compound/tau aggregate complex; (c) detecting the formation ofthe compound/tau aggregate complex; (d) collecting data for monitoringresidual disorder in the patient suffering from the disorder associatedwith tau aggregates who has been treated with the medicament; (e)optionally correlating the presence or absence of the compound/tauaggregate complex with the presence or absence of tau aggregate in thesample or specific body part or body area; and (f) optionally comparingthe amount of the compound/tau aggregate to a normal control value. 22.A method of collecting data for predicting responsiveness of a patientsuffering from a disorder associated with tau aggregates and beingtreated with a medicament comprising: (a) bringing a sample or aspecific body part or body area suspected to contain an tau aggregateinto contact with a compound as defined in claim 1, which compoundspecifically binds to the tau aggregate; (b) allowing the compound tobind to the tau aggregate to form a compound/tau aggregate complex; (c)detecting the formation of the compound/tau aggregate complex; (d)collecting data for predicting responsiveness of the patient sufferingfrom the disorder associated with tau aggregates and being treated withthe medicament; (e) optionally correlating the presence or absence ofthe compound/tau aggregate complex with the presence or absence of tauaggregate in the sample or specific body part or body area; and (f)optionally comparing the amount of the compound/tau aggregate to anormal control value.
 23. The method of claim 22, wherein the disorderassociated with tau aggregates is a tauopathy, Alzheimer's disease (AD),familial AD, Creutzfeldt-Jacob disease, dementia pugilistica, Down'sSyndrome, Gerstmann-Sträussler-Scheinker disease, inclusion-bodymyositis, prion protein cerebral amyloid angiopathy, traumatic braininjury, amyotrophic lateral sclerosis, Parkinsonism-dementia complex ofGuam, non-Guamanian motor neuron disease with neurofibrillary tangles,argyrophilic grain disease, corticobasal degeneration, diffuseneurofibrillary tangles with calcification, frontotemporal dementia withParkinsonism linked to chromosome 17, Hallervorden-Spatz disease,multiple system atrophy, Niemann-Pick disease type C,pallido-ponto-nigral degeneration, Pick's disease, progressivesubcortical gliosis, progressive supranuclear palsy (PSP), subacutesclerosing panencephalitis, tangle only dementia, postencephaliticParkinsonism, myotonic dystrophy, Tau panencephalopathy, AD-like withastrocytes, certain prion diseases (GSS with Tau), mutations in LRRK2,chronic traumatic encephalopathy, familial British dementia, familialDanish dementia, frontotemporal lobar degeneration, GuadeloupeanParkinsonism, neurodegeneration with brain iron accumulation,SLC9A6-related mental retardation, white matter tauopathy with globularglial inclusions, traumatic stress syndrome, epilepsy, Lewy bodydementia (LBD), hereditary cerebral hemorrhage with amyloidosis (Dutchtype), mild cognitive impairment (MCI), multiple sclerosis, Parkinson'sdisease, HIV-related dementia, adult onset diabetes, senile cardiacamyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primaryretinal degeneration, macular degeneration, optic nerve drusen, opticneuropathy, optic neuritis, and lattice dystrophy, or atypicalparkinsonism.
 24. The method of claim 23, wherein the tauopathy is a 3Rtauopathy or a 4R tauopathy.
 25. The method of claim 18, wherein thebody part or body area comprises the brain or the eye.
 26. The method ofclaim 18, wherein the detecting comprises positron emission tomographyimaging.